Biological methods for preparing adipic acid

ABSTRACT

The technology relates in part to biological methods for producing adipic acid and engineered microorganisms capable of such production.

RELATED PATENT APPLICATION

This patent application is a continuation application which claims thebenefit of international patent application no. PCT/US2010/040837 filedon Jul. 1, 2010, entitled BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID,naming Stephen Picataggio and Tom Beardslee as inventors and designatedby Attorney Docket No. VRD-1001-PC, which claims the benefit of U.S.provisional patent application No. 61/222,902 filed on Jul. 2, 2009,entitled BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID, naming StephenPicataggio as inventor and designated by Attorney Docket No.VRD-1001-PV. The entire content of the foregoing patent application isincorporated herein by reference, including, without limitation, alltext, tables and drawings.

FIELD

The technology relates in part to biological methods for producingadipic acid and engineered microorganisms capable of such production.

BACKGROUND

Microorganisms employ various enzyme-driven biological pathways tosupport their own metabolism and growth. A cell synthesizes nativeproteins, including enzymes, in vivo from deoxyribonucleic acid (DNA).DNA first is transcribed into a complementary ribonucleic acid (RNA)that comprises a ribonucleotide sequence encoding the protein. RNA thendirects translation of the encoded protein by interaction with variouscellular components, such as ribosomes. The resulting enzymesparticipate as biological catalysts in pathways involved in productionof molecules by the organism.

These pathways can be exploited for the harvesting of the naturallyproduced products. The pathways also can be altered to increaseproduction or to produce different products that may be commerciallyvaluable. Advances in recombinant molecular biology methodology allowresearchers to isolate DNA from one organism and insert it into anotherorganism, thus altering the cellular synthesis of enzymes or otherproteins. Such genetic engineering can change the biological pathwayswithin the host organism, causing it to produce a desired product.

Microorganic industrial production can minimize the use of causticchemicals and the production of toxic byproducts, thus providing a“clean” source for certain compounds.

SUMMARY

Provided herein are engineered microorganisms that produce six-carbonorganic molecules such as adipic acid, methods for manufacturing suchmicroorganisms and methods for using them to produce adipic acid andother six-carbon organic molecules.

Thus, provided herein in some embodiments are engineered microorganismscapable of producing adipic acid, or produce adipic acid, whichmicroorganisms comprise one or more altered activities selected from thegroup consisting of aldehyde dehydrogenase activity (e.g., 6-oxohexanoicacid dehydrogenase activity, omega oxo fatty acid dehydrogenaseactivity), fatty alcohol oxidase activity (e.g., 6-hydroxyhexanoic aciddehydrogenase activity, omega hydroxyl fatty acid dehydrogenaseactivity), hexanoate synthase activity and monooxygenase activity. Incertain embodiments, the microorganism comprises a genetic modificationthat adds or increases the aldehyde dehydrogenase activity (e.g.,6-oxohexanoic acid dehydrogenase activity, omega oxo fatty aciddehydrogenase activity), fatty alcohol oxidase activity (e.g.,6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity), hexanoate synthase activity, monooxygenaseactivity, monooxygenase reductase activity, fatty alcohol oxidaseactivity, acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoAhydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and/oracetyl-CoA C-acyltransferase activity. Also provided in someembodiments, are engineered microorganisms that produce adipic acid,which microorganisms comprise an altered monooxygenase activity.Provided also herein in some embodiments are engineered microorganismsthat include a genetic modification that reduces the acyl-CoA oxidaseactivity.

In some embodiments, an engineered microorganism includes a geneticmodification that includes multiple copies of a polynucleotide thatencodes a polypeptide having aldehyde dehydrogenase activity (e.g.,6-oxohexanoic acid dehydrogenase activity, omega oxo fatty aciddehydrogenase activity), fatty alcohol oxidase activity (e.g.,6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity), hexanoate synthase activity, monooxygenaseactivity, monooxygenase reductase activity, fatty alcohol oxidaseactivity, acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoAhydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and/oracetyl-CoA C-acyltransferase activity. In some embodiments, 2, 3, 4, 5,6, 7, 8, 9 or 10 or more copies of the particular polynucleotide arepresent in the microbe. In certain embodiments, an engineeredmicroorganism includes a heterologous promoter (and/or 5′UTR) infunctional connection with a polynucleotide that encodes a polypeptidehaving aldehyde dehydrogenase activity (e.g., 6-oxohexanoic aciddehydrogenase activity, omega oxo fatty acid dehydrogenase activity),fatty alcohol oxidase activity (e.g., 6-hydroxyhexanoic aciddehydrogenase activity, omega hydroxyl fatty acid dehydrogenaseactivity), hexanoate synthase activity, monooxygenase activity,monooxygenase reductase activity, fatty alcohol oxidase activity,acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoA hydrataseactivity, 3-hydroxyacyl-CoA dehydrogenase activity, and/or acetyl-CoAC-acyltransferase activity. In some embodiments, the promoter is a POX4or POX5 promoter or monooxygenase promoter from a yeast (e.g., Candidayeast strain (e.g., C. tropicalis strain)), or other promoter. Examplesof promoters that can be utilized are described herein. The promotersometimes is exogenous or endogenous with respect to the microbe.

Also provided herein is an engineered microorganism that produces adipicacid, where the microorganism includes an altered monooxygenaseactivity. In certain embodiments, an engineered microorganism comprisesa genetic modification that alters a monooxygenase activity. In someembodiments, an engineered microorganism includes a genetic modificationthat alters a monooxygenase activity selected from the group consistingof CYP52A15 activity, CYP52A16 activity, or CYP52A15 activity andCYP52A16 activity. In some embodiments, the monooxygenase activity isencoded by a CYP52A15 polynucleotide, a CYP52A16 polynucleotide, orCYP52A15 and CYP52A16 polynucleotides. In some embodiments, the geneticmodification increases monooxygenase activity. In certain embodiments,the genetic modification increases the copy number of an endogenouspolynucleotide that encodes a polypeptide having the monooxygenaseactivity (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of thepolynucleotide). In certain embodiments, an engineered microorganismcomprises a polynucleotide that includes a promoter (e.g., promoterand/or 5′UTR) and encodes a polypeptide having a monooxygenase activity.The promoter may be exogenous or endogenous with respect to the microbe.An engineered microorganism in certain embodiments comprises aheterologous polynucleotide encoding a polypeptide having monooxygenaseactivity. In related embodiments, the heterologous polynucleotide isfrom a yeast, such as a Candida yeast in certain embodiments (e.g., C.tropicalis).

In certain embodiments, an engineered microorganism comprises a geneticmodification that alters monooxygenase reductase activity. In someembodiments, the genetic modification increases the copy number of anendogenous polynucleotide that encodes a polypeptide havingmonooxygenase reductase activity (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore copies of the polynucleotide). An engineered microorganism incertain embodiments comprises a heterologous promoter (e.g., endogenousor exogenous promoter with respect to the microbe) in functionalconnection with a polynucleotide that encodes a polypeptide havingmonooxygenase reductase activity. In some embodiments, thepolynucleotide is from a yeast, and in certain embodiments the yeast isa Candida yeast (e.g., C. tropicalis).

An engineered microorganism in some embodiments comprises an alteredthioesterase activity. In some embodiments, an engineered microorganismcomprises a genetic modification that alters the thioesterase activity,and in certain embodiments, the engineered microorganism comprises agenetic alteration that adds or increases a thioesterase activity. Insome embodiments, the engineered microorganism comprises a heterologouspolynucleotide encoding a polypeptide having thioesterase activity.

An engineered microorganism in some embodiments comprises an alteredfatty alcohol oxidase activity. In some embodiments, an engineeredmicroorganism comprises a genetic modification that alters the fattyalcohol oxidase activity, and in certain embodiments, the engineeredmicroorganism comprises a genetic alteration that adds or increases afatty alcohol oxidase activity. In some embodiments, the geneticmodification increases the copy number of an endogenous polynucleotidethat encodes a polypeptide having fatty alcohol oxidase activity (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the polynucleotide). Anengineered microorganism in certain embodiments comprises a heterologouspromoter (e.g., endogenous or exogenous promoter with respect to themicrobe) in functional connection with a polynucleotide that encodes apolypeptide having fatty alcohol oxidase activity. In some embodiments,the engineered microorganism comprises a heterologous polynucleotideencoding a polypeptide having fatty alcohol oxidase activity. In someembodiments, the polynucleotide is from a yeast, and in certainembodiments the yeast is Candida (e.g., a C. tropicalis strain).

An engineered microorganism in some embodiments comprises an altered6-oxohexanoic acid dehydrogenase activity or an altered omega oxo fattyacid dehydrogenase activity. In some embodiments, an engineeredmicroorganism comprises a genetic modification that adds or increases6-oxohexanoic acid dehydrogenase activity or omega oxo fatty aciddehydrogenase activity, and in certain embodiments, an engineeredmicroorganism comprises a heterologous polynucleotide encoding apolypeptide having 6-oxohexanoic acid dehydrogenase activity or omegaoxo fatty acid dehydrogenase activity. In related embodiments, theheterologous polynucleotide sometimes is from a bacterium, such as anAcinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium in someembodiments.

An engineered microorganism in some embodiments comprises an altered6-hydroxyhexanoic acid dehydrogenase activity or an altered omegahydroxyl fatty acid dehydrogenase activity. In some embodiments, anengineered microorganism comprises a genetic modification that adds orincreases the 6-hydroxyhexanoic acid dehydrogenase activity or omegahydroxyl fatty acid dehydrogenase activity, and in certain embodiments,an engineered microorganism comprises a heterologous polynucleotideencoding a polypeptide having 6-hydroxyhexanoic acid dehydrogenaseactivity or omega hydroxyl fatty acid dehydrogenase activity. In relatedembodiments, the heterologous polynucleotide is from a bacterium, suchas an Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium insome embodiments.

An engineered microorganism in some embodiments comprises an alteredhexanoate synthase activity. In some embodiments, an engineeredmicroorganism comprises a genetic modification that alters hexanoatesynthase activity. In certain embodiments, an engineered microorganismincludes a genetic alteration that adds or increases hexanoate synthaseactivity. In some embodiments, an engineered microorganism comprises aheterologous polynucleotide encoding a polypeptide having hexanoatesynthase activity. In certain embodiments, the hexanoate synthaseactivity is provided by a polypeptide having hexanoate synthaseactivity. In certain embodiments, the hexanoate synthase activity isprovided by a polypeptide having hexanoate synthase subunit A activity,hexanoate synthase subunit B activity, or hexanoate synthase subunit Aactivity and hexanoate synthase subunit B activity. In some embodiments,the heterologous polynucleotide is from a fungus, such as an Aspergillusfungus in certain embodiments (e.g., A. parasiticus, A. nidulans).

In certain embodiments, an engineered microorganism comprises a geneticmodification that results in substantial (e.g., primary) hexanoate usageby monooxygenase activity. In related embodiments, the geneticmodification reduces a polyketide synthase activity.

An engineered microorganism in some embodiments is a non-prokaryoticorganism, and sometimes is a eukaryote. A eukaryote can be a yeast insome embodiments, such as a Candida yeast (e.g., C. tropicalis), forexample. In certain embodiments a eukaryote is a fungus, such as aYarrowia fungus (e.g., Y. lipolytica) or Aspergillus fungus (e.g., A.parasiticus or A. nidulans), for example.

In some embodiments, an engineered microorganism comprises a geneticmodification that reduces 6-hydroxyhexanoic acid conversion. In relatedembodiments, the genetic modification reduces 6-hydroxyhexanoic aciddehydrogenase activity or omega hydroxyl fatty acid dehydrogenaseactivity.

In certain embodiments, an engineered microorganism comprises a geneticmodification that reduces beta-oxidation activity, and in someembodiments, the genetic modification renders beta-oxidation activityundetectable (e.g., completely blocked beta-oxidation activity). Incertain embodiments, the genetic modification partially reducesbeta-oxidation activity. In some embodiments, an engineeredmicroorganism comprises a genetic modification that increasesomega-oxidation activity. In some embodiments, an engineeredmicroorganism comprises one or more genetic modifications that alter areverse activity in a beta oxidation pathway, an omega oxidationpathway, or a beta oxidation and omega oxidation pathway, therebyincreasing carbon flux through the respective pathways, due to thereduction in one or more reverse enzymatic activities.

A fatty acid-CoA derivative, or dicarboxylic acid-CoA derivative, can beconverted to a trans-2,3-dehydroacyl-CoA derivative by the activity ofacyl-CoA oxidase (e.g., also known as or referred to as acyl-CoAoxidoreductase and fatty acyl-coenzyme A oxidase), in many organisms. Insome embodiments, an engineered microorganism comprises a geneticmodification that alters the specificity of and/or reduces the activityof an acyl-CoA oxidase activity. In certain embodiments, the geneticmodification disrupts an acyl-CoA oxidase activity. In some embodiments,the genetic modification includes disrupting a polynucleotide thatencodes a polypeptide having an acyl-CoA oxidase activity. In certainembodiments, the genetic modification includes disrupting a promoterand/or 5′UTR in functional connection with a polynucleotide that encodesa polypeptide having the acyl-CoA oxidase activity. In some embodiments,the polypeptide having acyl-CoA activity is a POX polypeptide. Incertain embodiments, the POX polypeptide is a POX4 polypeptide, a POX5polypeptide, or a POX4 polypeptide and POX5 polypeptide. In certainembodiments, the genetic modification disrupts an acyl-CoA activity bydisrupting a POX4 nucleotide sequence, a POX5 nucleotide sequence, or aPOX4 and POX5 nucleotide sequence.

An engineered microorganism can include a heterologous polynucleotidethat encodes a polypeptide providing an activity described above, andthe heterologous polynucleotide can be from any suitable microorganism.Examples of microorganisms are described herein (e.g., Candida yeast,Saccharomyces yeast, Yarrowia yeast, Pseudomonas bacteria, Bacillusbacteria, Clostridium bacteria, Eubacterium bacteria and others includeMegasphaera bacteria.

Also provided in some embodiments are methods for manufacturing adipicacid, which comprise culturing an engineered microorganism describedherein under culture conditions in which the cultured microorganismproduces adipic acid. In some embodiments, the host microorganism fromwhich the engineered microorganism is generated does not produce adetectable amount of adipic acid. In certain embodiments, the cultureconditions comprise fermentation conditions, introduction of biomass,introduction of glucose, introduction of a paraffin (e.g., plant orpetroleum based, such as hexane or coconut oil, for example) and/orcombinations thereof. In some embodiments, the adipic acid is producedwith a yield of greater than about 0.3 grams per gram of glucose added.In related embodiments, a method comprises purifying the adipic acidfrom the cultured microorganisms and or modifying the adipic acid,thereby producing modified adipic acid. In certain embodiments, a methodcomprises placing the cultured microorganisms, the adipic acid or themodified adipic acid in a container, and optionally, shipping thecontainer.

Provided also in certain embodiments are methods for manufacturing6-hydroxyhexanoic acid, which comprise culturing an engineeredmicroorganism described herein under culture conditions in which thecultured microorganism produces 6-hydroxyhexanoic acid. In someembodiments, the host microorganism from which the engineeredmicroorganism is generated does not produce a detectable amount of6-hydroxyhexanoic acid. In certain embodiments, the culture conditionscomprise fermentation conditions, introduction of biomass, introductionof glucose, and/or introduction of hexane. In some embodiments, the6-hydroxyhexanoic acid is produced with a yield of greater than about0.3 grams per gram of glucose added. In related embodiments, a methodcomprises purifying the 6-hydroxyhexanoic acid from the culturedmicroorganisms and or modifying the 6-hydroxyhexanoic acid, therebyproducing modified 6-hydroxyhexanoic acid. In certain embodiments, amethod comprises placing the cultured microorganisms, the6-hydroxyhexanoic acid or the modified 6-hydroxyhexanoic acid in acontainer, and optionally, shipping the container.

Also provided in some embodiments are methods for preparing anengineered microorganism that produces adipic acid, which comprise: (a)introducing a genetic modification to a host organism that adds orincreases monooxygenase activity, thereby producing engineeredmicroorganisms having detectable and/or increased monooxygenaseactivity; and (b) selecting for engineered microorganisms that produceadipic acid. Provided also herein in some embodiments are methods forpreparing an engineered microorganism that produces adipic acid, whichcomprise: (a) culturing a host organism with hexane as a nutrientsource, thereby producing engineered microorganisms having detectablemonooxygenase activity; and (b) selecting for engineered microorganismsthat produce adipic acid. In some embodiments the monooxygenase activityis incorporation of a hydroxyl moiety into a six-carbon molecule, and incertain embodiments, the six-carbon molecule is hexanoate. In relatedembodiments, a method comprises selecting the engineered microorganismsthat have a detectable amount of the monooxygenase activity. In someembodiments, a method comprises introducing a genetic modification thatadds or increases a hexanoate synthase activity, thereby producingengineered microorganisms, and selecting for engineered microorganismshaving detectable and/or increased hexanoate synthase activity. Inrelated embodiments, the genetic modification encodes a polypeptidehaving a hexanoate synthase subunit A activity, a hexanoate synthasesubunit B activity, or a hexanoate synthase subunit A activity and ahexanoate synthase subunit B activity.

In some embodiments, a method comprises introducing a geneticmodification that adds or increases an aldehyde dehydrogenase activity(e.g., 6-oxohexanoic acid dehydrogenase activity, omega oxo fatty aciddehydrogenase), thereby producing engineered microorganisms, andselecting for engineered microorganisms having detectable and/orincreased 6-oxohexanoic acid dehydrogenase activity or omega oxo fattyacid dehydrogenase relative to the host microorgansim. In certainembodiments, a method for preparing microorganisms that produce adipicacid includes selecting for engineered microorganisms having one or moredetectable and/or increased activities selected from the groupconsisting of an aldehyde dehydrogenase activity (e.g., 6-oxohexanoicacid dehydrogenase activity, omega oxo fatty acid dehydrogenase), fattyalcohol oxidase activity (e.g., 6-hydroxyhexanoic acid dehydrogenaseactivity, omega hydroxyl-fatty acid dehydrogenase), hexanoate synthaseactivity, monooxygenase activity, monooxygenase reductase activity,fatty alcohol oxidase activity, acyl-CoA ligase activity, acyl-CoAoxidase activity, enoyl-CoA hydratase activity, 3-hydroxyacyl-CoAdehydrogenase activity, and/or acetyl-CoA C-acyltransferase activity.

In certain embodiments, a method comprises introducing a geneticmodification that adds or increases a fatty alcohol oxidase activity(e.g., 6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxylfatty acid dehydrogenase activity) thereby producing engineeredmicroorganisms, and selecting for engineered microorganisms having adetectable and/or increased 6-hydroxyhexanoic acid dehydrogenaseactivity or omega hydroxyl fatty acid dehydrogenase activity relative tothe host microorganism. In some embodiments, a method comprisesintroducing a genetic modification that adds or increases a thioesteraseactivity, thereby producing engineered microorganisms, and selecting forengineered microorganisms having a detectable and/or increasedthioesterase activity relative to the host microorganism.

In certain embodiments, a method comprises introducing a geneticmodification that reduces 6-hydroxyhexanoic acid conversion, therebyproducing engineered microorganisms, and selecting for engineeredmicroorganisms having reduced 6-hydroxyhexanoic acid conversion relativeto the host microorganism. In some embodiments, a method comprisesintroducing a genetic modification that reduces beta-oxidation activity,thereby producing engineered microorganisms, and selecting forengineered microorganisms having reduced beta-oxidation activityrelative to the host microorganism. In certain embodiments, a methodcomprises introducing a genetic modification that results in substantialhexanoate usage by the monooxygenase activity, thereby producingengineered microorganisms, and selecting for engineered microorganismsin which substantial hexanoate usage is by the monooxygenase activityrelative to the host microorganism. In some embodiments, a methodcomprises introducing a genetic modification that increasesomega-oxidation activity, thereby producing engineered microorganisms,and selecting for engineered microorganisms having increasedomega-oxidation activity relative to the host microorganism.

Provided also herein in certain embodiments are methods for preparing amicroorganism that produces adipic acid, which comprise: (a) introducingone or more genetic modifications to a host organism that add orincrease one or more activities selected from the group consisting of6-oxohexanoic acid dehydrogenase activity, omega oxo fatty aciddehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenase activity,omega hydroxyl fatty acid dehydrogenase activity, hexanoate synthaseactivity, monooxygenase activity, monooxygenase reductase activity,fatty alcohol oxidase activity, acyl-CoA ligase activity, acyl-CoAoxidase activity, enoyl-CoA hydratase activity, 3-hydroxyacyl-CoAdehydrogenase activity, and/or acetyl-CoA C-acyltransferase activity,thereby producing engineered microorganisms, and (b) selecting forengineered microorganisms that produce adipic acid. In some embodiments,a method comprises selecting for engineered microorganisms having one ormore detectable and/or increased activities selected from the groupconsisting of 6-oxohexanoic acid dehydrogenase activity,6-hydroxyhexanoic acid dehydrogenase activity, hexanoate synthaseactivity, monooxygenase activity, monooxygenase reductase activity,fatty alcohol oxidase activity, acyl-CoA ligase activity, acyl-CoAoxidase activity, enoyl-CoA hydratase activity, 3-hydroxyacyl-CoAdehydrogenase activity, and/or acetyl-CoA C-acyltransferase activity,relative to the host microorganism.

In certain embodiments, a method comprises introducing a geneticmodification that reduces 6-hydroxyhexanoic acid conversion, therebyproducing engineered microorganisms, and selecting for engineeredmicroorganisms having reduced 6-hydroxyhexanoic acid conversion relativeto the host microorganism. In some embodiments, a method comprisesintroducing a genetic modification that reduces beta-oxidation activity,thereby producing engineered microorganisms, and selecting forengineered microorganisms having reduced beta-oxidation activityrelative to the host microorganism. In certain embodiments, a methodcomprises introducing a genetic modification that results in substantialhexanoate usage by the monooxygenase activity, thereby producingengineered microorganisms, and selecting for engineered microorganismsin which substantial hexanoate usage is by the monooxygenase activityrelative to the host microorganism.

Also provided in some embodiments are methods for preparing amicroorganism that produces 6-hydroxyhexanoic acid, which comprise: (a)introducing one or more genetic modifications to a host organism thatadd or increase one or more activities selected from the groupconsisting of 6-oxohexanoic acid dehydrogenase activity, hexanoatesynthase activity, monooxygenase activity, monooxygenase reductaseactivity, fatty alcohol oxidase activity, acyl-CoA ligase activity,acyl-CoA oxidase activity, enoyl-CoA hydratase activity,3-hydroxyacyl-CoA dehydrogenase activity, and/or acetyl-CoAC-acyltransferase activity, thereby producing engineered microorganisms,(b) introducing a genetic modification to the host organism that reduces6-hydroxyhexanoic acid conversion, and (c) selecting for engineeredmicroorganisms that produce 6-hydroxyhexanoic acid. In certainembodiments, a method comprises selecting for engineered microorganismshaving reduced 6-hydroxyhexanoic acid conversion relative to the hostmicroorganism. In some embodiments, a method comprises selecting forengineered microorganisms having one or more detectable and/or increasedactivities selected from the group consisting of aldehyde dehydrogenaseactivity (e.g., 6-oxohexanoic acid dehydrogenase activity, omega oxofatty acid dehydrogenase activity), fatty alcohol oxidase activity(e.g., 6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxylfatty acid dehydrogenase activity), hexanoate synthase activity,monooxygenase activity, monooxygenase reductase activity, fatty alcoholoxidase activity, acyl-CoA ligase activity, acyl-CoA oxidase activity,enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity,and/or acetyl-CoA C-acyltransferase activity, relative to the hostmicroorganism. In certain embodiments, a method comprises introducing agenetic modification that reduces beta-oxidation activity, therebyproducing engineered microorganisms, and selecting for engineeredmicroorganisms having reduced beta-oxidation activity relative to thehost microorganism. In some embodiments, a method comprises introducinga genetic modification that results in substantial hexanoate usage bythe monooxygenase activity, thereby producing engineered microorganisms,and selecting for engineered microorganisms in which substantialhexanoate usage is by the monooxygenase activity relative to the hostmicroorganism.

Also provided are methods that include contacting an engineeredmicroorganism with a feedstock including one or more polysaccharides,wherein the engineered microorganism includes: (a) a genetic alterationthat blocks beta oxidation activity, and (b) a genetic alteration thatadds or increases a monooxygenase activity or a genetic alteration thatadds or increases a hexanoate synthetase activity, and culturing theengineered microorganism under conditions in which adipic acid isproduced. In some embodiments, the engineered microorganism comprises agenetic alteration that adds or increases hexanoate synthetase activity.In certain embodiments, the engineered microorganism comprises aheterologous polynucleotide encoding a polypeptide having hexanoatesynthase subunit A activity, and in some embodiments the engineeredmicroorganism comprises a heterologous polynucleotide encoding apolypeptide having hexanoate synthase subunit B activity. In certainembodiments, the heterologous polynucleotide independently is selectedfrom a fungus. In some embodiments, the fungus is an Aspergillus fungus,and in certain embodiments the Aspergillus fungus is A. parasiticus. Insome embodiments, wherein the microorganism is a Candida yeast, and incertain embodiments, the microorganism is a C. tropicalis strain.

Provided also are methods that include contacting an engineeredmicroorganism with a feedstock comprising one or more paraffins, whereinthe engineered microorganism comprises a genetic alteration thatpartially blocks beta oxidation activity and culturing the engineeredmicroorganism under conditions in which adipic acid is produced. Incertain embodiments, the microorganism comprises a genetic alterationthat increases a monooxygenase activity. In some embodiments, themicroorganism is a Candida yeast, and in certain embodiments, themicroorganism is a C. tropicalis strain.

In some embodiments, the genetic alteration that increases monooxygenaseactivity comprises a genetic alteration that increases Cytochrome P450reductase activity. In certain embodiments, the genetic alterationincreases the number of copies of a polynucleotide that encodes apolypeptide having the Cytochrome P450 reductase activity. In someembodiments, the genetic alteration places a promoter and/or 5′UTR infunctional connection with a polynucleotide that encodes a polypeptidehaving the Cytochrome P450 reductase activity. In certain embodiments,the monooxygenase activity is a CYP52A15 activity, CYP52A16 activity, ora CYP52A15 activity and CYP52A16 activity. In some embodiments, thegenetic alteration increases the number of copies of a polynucleotidethat encodes a polypeptide having the monooxygenase activity. In certainembodiments, the genetic alteration places a promoter and/or 5′UTR infunctional connection with a polynucleotide that encodes a polypeptidehaving the monooxygenase activity.

In certain embodiments, the genetic alteration that blocks betaoxidation activity disrupts acyl-CoA oxidase activity. In someembodiments, the genetic alteration disrupts POX4 and/or POX5 activity.In certain embodiments, the genetic alteration disrupts a polynucleotidethat encodes a polypeptide having the acyl-CoA oxidase activity. In someembodiments, the genetic alteration disrupts a promoter and/or 5′UTR infunctional connection with a polynucleotide that encodes a polypeptidehaving the acyl-CoA oxidase activity.

In some embodiments, the feedstock comprises a 6-carbon sugar. Incertain embodiments, the feedstock comprises a 5-carbon sugar. Incertain embodiments, the adipic acid is produced at a level of about 80%or more of theoretical yield. In some embodiments, the amount of adipicacid produced is detected. In certain embodiments, the adipic acidproduced is isolated (e.g., partially or completely purified). In someembodiments, the culture conditions comprise fermenting the engineeredmicroorganism.

Provided also herein are engineered microorganisms in contact with afeedstock. In some embodiments, the feedstock includes a saccharide. Incertain embodiments, the saccharide is a monosaccharide, polysaccharide,or a mixture of a monosaccharide and polysaccharide. In someembodiments, the feedstock includes a paraffin. In certain embodiments,the paraffin is a saturated paraffin, unsaturated paraffin, substitutedparaffin, branched paraffin, linear paraffin, or combination thereof.

In some embodiments, the paraffin includes about 1 to about 60 carbonatoms (e.g., between about 1 carbon atom, about 2 carbon atoms, about 3carbon atoms, about 4 carbon atoms, about 5 carbon atoms, about 6 carbonatoms, about 7 carbon atoms, about 8 carbon atoms, about 9 carbon atoms,about 10 carbon atoms, about 12 carbon atoms, about 14 carbon atoms,about 16 carbon atoms, about 18 carbon atoms, about 20 carbon atoms,about 22 carbon atoms, about 24 carbon atoms, about 26 carbon atoms,about 28 carbon atoms, about 30 carbon atoms, about 32 carbon atoms,about 34 carbon atoms, about 36 carbon atoms, about 38 carbon atoms,about 40 carbon atoms, about 42 carbon atoms, about 44 carbon atoms,about 46 carbon atoms, about 48 carbon atoms, about 50 carbon atoms,about 52 carbon atoms, about 54 carbon atoms, about 56 carbon atoms,about 58 carbon atoms and about 60 carbon atoms). In certainembodiments, the paraffin is in a mixture of paraffins. In someembodiments, the paraffins in the mixture of paraffins have a meannumber of carbon atoms of about 8 carbon atoms to about 18 carbon atoms(e.g., about 8 carbon atoms, about 9 carbon atoms, about 10 carbonatoms, about 11 carbon atoms, about 12 carbon atoms, about 13 carbonatoms, about 14 carbon atoms, about 15 carbon atoms, about 16 carbonatoms, about 17 carbon atoms or about 18 carbon atoms). In certainembodiments, the paraffin is in a wax, and in some embodiments, theparaffin is in an oil. In certain embodiments, the paraffin is from apetroleum product, and in some embodiments, the petroleum product is apetroleum distillate. In certain embodiments, the paraffin is from aplant or plant product.

Also provided herein, is an isolated polynucleotide selected from thegroup including a polynucleotide having a nucleotide sequence 96% ormore (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%)identical to the nucleotide sequence of SEQ ID NO: 1, a polynucleotidehaving a nucleotide sequence that encodes a polypeptide of SEQ ID NO: 8,and a polynucleotide having a portion of a nucleotide sequence 96% ormore identical to the nucleotide sequence of SEQ ID NO: 1 and encodes apolypeptide having fatty alcohol oxidase activity.

Also provided herein, is an isolated polynucleotide selected from thegroup including a polynucleotide having a nucleotide sequence 98% ormore (e.g., 98% or more, 99% or more, or 100%) identical to thenucleotide sequence of SEQ ID NO: 2, a polynucleotide having anucleotide sequence that encodes a polypeptide of SEQ ID NO:10, and apolynucleotide having a portion of a nucleotide sequence 98% or moreidentical to the nucleotide sequence of SEQ ID NO: 2 and encodes apolypeptide having fatty alcohol oxidase activity.

Also provided herein, is an isolated polynucleotide selected from thegroup including a polynucleotide having a nucleotide sequence 95% ormore (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% ormore, or 100%) identical to the nucleotide sequence of SEQ ID NO: 3, apolynucleotide having a nucleotide sequence that encodes a polypeptideof SEQ ID NO: 9, and a polynucleotide having a portion of a nucleotidesequence 95% or more identical to the nucleotide sequence of SEQ ID NO:3 and encodes a polypeptide having fatty alcohol oxidase activity.

Also provided herein, is an isolated polynucleotide selected from thegroup including a polynucleotide having a nucleotide sequence 83% ormore (e.g., 83% or more, 84% or more, 85% or more, 86% or more, 87% ormore, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more,93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more, or 100%) identical to the nucleotide sequence of SEQID NO: 4, a polynucleotide having a nucleotide sequence that encodes apolypeptide of SEQ ID NO: 11, and a polynucleotide having a portion of anucleotide sequence 83% or more identical to the nucleotide sequence ofSEQ ID NO: 3 and encodes a polypeptide having fatty alcohol oxidaseactivity.

Also provided herein, is an isolated polynucleotide selected from thegroup including a polynucleotide having a nucleotide sequence 82% ormore (e.g., 82% or more, 83% or more, 84% or more, 85% or more, 86% ormore, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more, or 100%) identical to the nucleotidesequence of SEQ ID NO: 5, a polynucleotide having a nucleotide sequencethat encodes a polypeptide of SEQ ID NO: 12, and a polynucleotide havinga portion of a nucleotide sequence 82% or more identical to thenucleotide sequence of SEQ ID NO: 3 and encodes a polypeptide havingfatty alcohol oxidase activity.

In certain embodiments, an expression vector includes a polynucleotidesequence of SEQ ID NOs: 1 to 5. In some embodiments, an integrationvector includes a polynucleotide sequence of SEQ ID NOs: 1 to 5. Incertain embodiments, a microorganism includes an expression vector, anintegration vector, or an expression vector and an integration vectorthat includes a polynucleotide sequence of SEQ ID NOs: 1 to 5. In someembodiments, a culture includes a microorganism that includes anexpression vector, an integration vector, or an expression vector and anintegration vector that includes a polynucleotide sequence of SEQ IDNOs: 1 to 5. In certain embodiments, a fermentation device includes amicroorganism that includes an expression vector, an integration vector,or an expression vector and an integration vector that includes apolynucleotide sequence of SEQ ID NOs: 1 to 5. Also provided herein is apolypeptide encoded by a polynucleotide sequence of SEQ ID NOs: 1 to 5or produced by an expression vector that includes a polynucleotidesequence of SEQ ID NOs: 1 to 5. Provided also herein is an antibody thatspecifically binds to a polypeptide encoded by a polynucleotide sequenceof SEQ ID NOs: 1 to 5 or produced by an expression vector that includesa polynucleotide sequence of SEQ ID NOs: 1 to 5.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 depicts a metabolic pathway for making adipic acid. The pathwaycan be engineered into a eukaryotic microorganism to generate amicroorganism capable of producing adipic acid.

FIG. 2 depicts an embodiment for a method of generating an adipic acidproducing microorganism. The method comprises expressing one or moregenes catalyzing the omega oxidation of fatty acids to dicarboxylicacids in a host microorganism that produces hexanoate. In the methoddepicted, the host organism, for example A. parasiticus or A. nidulans,endogenously includes HEXA and HEXB (or STCJ and STCK) genes. In oneembodiment the method comprises knocking out or otherwise disabling thegene coding for diversion of hexanoate into an endogenous pathway suchas mycotoxin production. Certain embodiments of the method furthercomprise inserting a heterologous cytochrome P450 gene. Some embodimentsof the method comprise growing the culture on hexane and screening forincreased P450 expression. The copy number of hexanoate induced P450 mayin certain embodiments be increased. In some embodiments themicroorganism may be altered to increase the flux of six carbonsubstrate through the final two oxidation steps.

FIG. 3 depicts an embodiment for a method of generating an adipic acidproducing organism. The method comprises expressing one or more genesencoding hexanoate synthase in a host microorganism that producesdicarboxylic acids via an omega-oxidation pathway. Such microorganismsmay include, without limitation, C. tropicalis and C. maltosa. Asdepicted, the method comprises inserting HEXA and HEXB genes into thehost microorganism. The genes may be isolated from Aspergillus, oranother appropriate organism. In some embodiments, the genes aresynthesized from an alternative sequence as described herein to producethe amino acid sequence of the donor microorganism enzyme through anon-standard translation mechanism of C. tropicalis. In some embodimentsthe method comprises inserting a heterologous cytochrome P450 gene intothe host organism. In certain embodiments the microorganism may bealtered to increase the flux of a six-carbon substrate through the finaltwo oxidation steps.

FIG. 4 depicts an embodiment of a method for generating an adipic acidproducing organism. The method comprises expressing one or more genesencoding hexanoate synthase in a host microorganism that producesdicarboxylic acids via an omega-oxidation pathway. The microorganismscan include, without limitation, C. tropicalis and C. maltosa. In someembodiments, the method comprises growing a host microorganism on hexaneand screening for increased P450 expression. In certain embodiments,copy number of hexane-induced P450 may be increased. HEXA and HEXB genesmay be inserted into the host microorganism. In certain embodiments, thehost microorganism may be altered to increase the flux of a six-carbonsubstrate through the final two oxidation steps.

FIG. 5 depicts a plasmid diagram for inserting Aspergillus hexanoatesynthase genes HEXA and HEXB into C. tropicalis or Y. lipolytica.

FIG. 6 depicts a plasmid diagram for inserting a heterologous cytochromeP450 monooxygenase gene and cytochrome P450 reductase gene into C.tropicalis or Y. lipolytica.

FIG. 7 depicts a plasmid diagram for inserting a heterologous cytochromeP450 monooxygenase gene and cytochrome P450 reductase gene into A.parasiticus or A. nidulans.

FIG. 8 depicts a system for biological production of a target product.As depicted, a fermenter is populated with microorganisms engineered fortarget product production. A flexible feedstock supplies the fermenterwith an energy and nutrition source for the microorganisms. In someembodiments the feedstock comprises a sugar. In certain embodiments thefeedstock comprises fatty acids. The feedstock may also include biomass,industrial waste products and other sources of carbon. Vitamins,minerals, enzymes and other growth or production enhancers may be addedto the feedstock. In certain embodiments the fermentation producesadipic acid. The fermentation process may produce other novel chemicals.

FIG. 9 depicts a metabolic pathway for making adipic acid fromsaccharide or polysaccharide carbon sources, similar to the pathwaydepicted in FIG. 1, with additional activities that aid in metabolismof, or enhance metabolism of, pathway intermediates, thereby potentiallyincreasing the yield of adipic acid. The additional activities are amonooxygenase reductase activity (cytochrome P450 reductase or CPR) anda fatty alcohol oxidase activity (FAO). Part, or all, of the pathway canbe engineered into a eukaryotic microorganism to generate amicroorganism capable of producing adipic acid.

FIG. 10 depicts a non-limiting example of a metabolic pathway for makingadipic acid from paraffins, fats, oils, fatty acids or dicarboxylicacids, as described in FIG. 2. Part, or all, of the pathway can beengineered (e.g., added, altered to increase or decrease copy number, orincrease or decrease promoter activity, depending on the desired effect)into a microorganism, depending on the activities already present in thehost organism, to generate a microorganism capable of producing adipicacid.

FIGS. 11A and 11B depict omega and beta oxidation pathways useful forproducing adipic acid from various carbon sources. Adipic acid can beproduced from paraffins, fats, oils and intermediates of sugarmetabolism, using omega oxidation, as shown in FIG. 11A. Adipic acidalso can be produced from long chain fatty acids or dicarboxylic acidsusing beta oxidation, as shown in FIG. 11A. FIG. 11B shows a commonintermediate from the metabolism of fats and sugars entering the omegaoxidation pathway to ultimately produce adipic acid.

FIG. 12 shows results of immunodetection of 6×His-tagged proteinsexpressed in S. cerevisiae BY4742. Strains sAA061, sAA140, sAA141,sAA142 contain 6×His-tagged HEXA and HEXB proteins. Strain sAA144contains 6×His-tagged STCJ and STCK proteins. Strain sAA048 containsonly vectors p425GPD and p426GPD.

FIG. 13 shows results of immunodetection of 6×His-tagged proteinsexpressed in either S. cerevisiae (sAA144) or in C. tropicalis (sAA103,sAA270, sAA269). 6×His tagged HEXA and HEXB expressed in strains sAA269and sAA270 are indicated with arrows. 6×His tagged STCJ and STCK fromstrain sAA144 were included as a positive control. Strain sAA103 is theparent strain for sAA269 and sAA270 and does not contain integratedvectors for the expression of 6×His-tagged HEXA and HEXB.

FIG. 14 shows results of RT-PCR from cultures of C. tropicalis strainsAA003 exposed to glucose only (Glc), hexane only (Hex), or hexanoicacid only (HA). PCR products of A15 and A16 alleles show hexane andhexanoic acid specific induction.

FIGS. 15A-15C illustrate results of acyl-CoA oxidase (POX) enzymaticactivity assays on substrates of various carbon lengths, using acyl-CoAenzyme preparations from Candida tropicalis strains with no POX genesdisrupted (see FIG. 15A), POX4 genes disrupted (see FIG. 15C) or POX5genes disrupted (see FIG. 15B). Experimental results and conditions aregiven in the Detailed Description and Examples sections.

FIGS. 16-34 illustrate various plasmids for cloning, expression, orintegration of various activities described herein, into a host organismor engineered organism.

FIG. 16 depicts a plasmid diagram for inserting a heterologous HEXA geneinto S. cerevisiae.

FIG. 17 depicts a plasmid diagram for inserting a heterologous HEXB geneinto S. cerevisiae.

FIG. 18 depicts a plasmid diagram for inserting a heterologousHEXA-6×His gene into S. cerevisiae.

FIG. 19 depicts a plasmid diagram for inserting a heterologousHEXB-6×His gene into S. cerevisiae.

FIG. 20 depicts a plasmid diagram for inserting a heterologous STCJ geneinto S. cerevisiae.

FIG. 21 depicts a plasmid diagram for inserting a heterologous STCK geneinto S. cerevisiae.

FIG. 22 depicts a plasmid diagram for inserting a heterologousSTCJ-6×His gene into S. cerevisiae.

FIG. 23 depicts a plasmid diagram for inserting a heterologousSTCK-6×His gene into S. cerevisiae.

FIG. 24 depicts a plasmid diagram for inserting a heterologousalternative genetic code (AGC) HEXA gene into C. tropicalis.

FIG. 25 depicts a plasmid diagram for inserting a heterologous AGC-HEXBgene into C. tropicalis.

FIG. 26 depicts a plasmid diagram for inserting a heterologousAGC-HEXA-6×His gene into C. tropicalis.

FIG. 27 depicts a plasmid diagram for inserting a heterologousAGC-HEXB-6×His gene into C. tropicalis.

FIG. 28 depicts a diagram of a plasmid used for cloning the POX5 genefrom C. tropicalis.

FIG. 29 depicts a diagram of a plasmid used for cloning the POX4 genefrom C. tropicalis.

FIG. 30 illustrates a plasmid constructed for use of URA selection in C.tropicalis.

FIG. 31 depicts a plasmid containing the PGK promoter and terminatorfrom C. tropicalis.

FIG. 32 depicts a plasmid used for integration of the CPR gene in C.tropicalis.

FIG. 33 depicts a plasmid used for integration of the CYP52A15 gene inC. tropicalis.

FIG. 34 depicts a plasmid used for integration of the CYP52A16 gene inC. tropicalis.

DETAILED DESCRIPTION

Adipic acid is a six-carbon organic molecule that is a chemicalintermediate in manufacturing processes used to make certain polyamides,polyurethanes and plasticizers, all of which have wide applications inproducing items such as carpets, coatings, adhesives, elastomers, foodpackaging, and lubricants, for example. Some large-scale processes formaking adipic acid include (i) liquid phase oxidation of ketone alcoholoil (KA oil); (ii) air oxidation/hydration of cyclohexane with boricacid to make cyclohexanol, followed by oxidation with nitric acid; and(iii) hydrocyanation of butadiene to a pentenenitrile mixture, followedby hydroisomerization of adiponitrile, followed by hydrogenation. Eachof the latter processes requires use of noxious chemicals and/orsolvents, some require high temperatures, and all require significantenergy input. In addition, some of the processes emit toxic byproducts(such as nitrous oxide) and give rise to environmental concerns.

Provided herein are methods for producing adipic acid and other organicchemical intermediates using biological systems. Such production systemsmay have significantly less environmental impact and could beeconomically competitive with current manufacturing systems. Thus,provided herein are methods for manufacturing adipic acid by engineeredmicroorganisms. In some embodiments microorganisms are engineered tocontain at least one heterologous gene encoding an enzyme, where theenzyme is a member of a novel pathway engineered into the microorganism.In certain embodiments, an organism may be selected for elevatedactivity of a native enzyme.

Microorganisms

A microorganism selected often is suitable for genetic manipulation andoften can be cultured at cell densities useful for industrial productionof a target product. A microorganism selected often can be maintained ina fermentation device.

The term “engineered microorganism” as used herein refers to a modifiedmicroorganism that includes one or more activities distinct from anactivity present in a microorganism utilized as a starting point(hereafter a “host microorganism”). An engineered microorganism includesa heterologous polynucleotide in some embodiments, and in certainembodiments, an engineered organism has been subjected to selectiveconditions that alter an activity, or introduce an activity, relative tothe host microorganism. Thus, an engineered microorganism has beenaltered directly or indirectly by a human being. A host microorganismsometimes is a native microorganism, and at times is a microorganismthat has been engineered to a certain point.

In some embodiments an engineered microorganism is a single cellorganism, often capable of dividing and proliferating. A microorganismcan include one or more of the following features: aerobe, anaerobe,filamentous, non-filamentous, monoploid, dipoid, auxotrophic and/ornon-auxotrophic. In certain embodiments, an engineered microorganism isa prokaryotic microorganism (e.g., bacterium), and in certainembodiments, an engineered microorganism is a non-prokaryoticmicroorganism. In some embodiments, an engineered microorganism is aeukaryotic microorganism (e.g., yeast, fungi, amoeba).

Any suitable yeast may be selected as a host microorganism, engineeredmicroorganism or source for a heterologous polynucleotide. Yeastinclude, but are not limited to, Yarrowia yeast (e.g., Y. lipolytica(formerly classified as Candida lipolytica)), Candida yeast (e.g., C.revkaufi, C. pulcherrima, C. tropicalis, C. utilis), Rhodotorula yeast(e.g., R. glutinus, R. graminis), Rhodosporidium yeast (e.g., R.toruloides), Saccharomyces yeast (e.g., S. cerevisiae, S. bayanus, S.pastorianus, S. carlsbergensis), Cryptococcus yeast, Trichosporon yeast(e.g., T. pullans, T. cutaneum), Pichia yeast (e.g., P. pastoris) andLipomyces yeast (e.g., L. starkeyii, L. lipoferus). In some embodiments,a yeast is a Y. lipolytica strain that includes, but is not limited to,ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982 and LGAM S(7)1strains (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9(2002)). In certain embodiments, a yeast is a C. tropicalis strain thatincludes, but is not limited to, ATCC20336, ATCC20913, SU-2(ura3-/ura3-), ATCC20962, H5343 (beta oxidation blocked; U.S. Pat. No.5,648,247) strains.

Any suitable fungus may be selected as a host microorganism, engineeredmicroorganism or source for a heterologous polynucleotide. Non-limitingexamples of fungi include, but are not limited to, Aspergillus fungi(e.g., A. parasiticus, A. nidulans), Thraustochytrium fungi,Schizochytrium fungi and Rhizopus fungi (e.g., R. arrhizus, R. oryzae,R. nigricans). In some embodiments, a fungus is an A. parasiticus strainthat includes, but is not limited to, strain ATCC24690, and in certainembodiments, a fungus is an A. nidulans strain that includes, but is notlimited to, strain ATCC38163.

Any suitable prokaryote may be selected as a host microorganism,engineered microorganism or source for a heterologous polynucleotide. AGram negative or Gram positive bacteria may be selected. Examples ofbacteria include, but are not limited to, Bacillus bacteria (e.g., B.subtilis, B. megaterium), Acinetobacter bacteria, Norcardia baceteria,Xanthobacter bacteria, Escherichia bacteria (e.g., E. coli (e.g.,strains DH10B, Stbl2, DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 andccdA-over (e.g., U.S. application Ser. No. 09/518,188))), Streptomycesbacteria, Erwinia bacteria, Klebsiella bacteria, Serratia bacteria(e.g., S. marcessans), Pseudomonas bacteria (e.g., P. aeruginosa),Salmonella bacteria (e.g., S. typhimurium, S. typhi), Megasphaerabacteria (e.g., Megasphaera elsdenii). Bacteria also include, but arenot limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria(e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema bacteria(e.g., C. gigateum)), green sulfur bacteria (e.g., Chlorobium bacteria(e.g., C. limicola), Pelodictyon bacteria (e.g., P. luteolum), purplesulfur bacteria (e.g., Chromatium bacteria (e.g., C. okenii)), andpurple non-sulfur bacteria (e.g., Rhodospirillum bacteria (e.g., R.rubrum), Rhodobacter bacteria (e.g., R. sphaeroides, R. capsulatus), andRhodomicrobium bacteria (e.g., R. vanellii)).

Cells from non-microbial organisms can be utilized as a hostmicroorganism, engineered microorganism or source for a heterologouspolynucleotide. Examples of such cells, include, but are not limited to,insect cells (e.g., Drosophila (e.g., D. melanogaster), Spodoptera(e.g., S. frugiperda Sf9 or Sf21 cells) and Trichoplusa (e.g., High-Fivecells); nematode cells (e.g., C. elegans cells); avian cells; amphibiancells (e.g., Xenopus laevis cells); reptilian cells; and mammalian cells(e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanomaand HeLa cells).

Microorganisms or cells used as host organisms or source for aheterologous polynucleotide are commercially available. Microorganismsand cells described herein, and other suitable microorganisms and cellsare available, for example, from Invitrogen Corporation, (Carlsbad,Calif.), American Type Culture Collection (Manassas, Va.), andAgricultural Research Culture Collection (NRRL; Peoria, Ill.).

Host microorganisms and engineered microorganisms may be provided in anysuitable form. For example, such microorganisms may be provided inliquid culture or solid culture (e.g., agar-based medium), which may bea primary culture or may have been passaged (e.g., diluted and cultured)one or more times. Microorganisms also may be provided in frozen form ordry form (e.g., lyophilized). Microorganisms may be provided at anysuitable concentration.

Carbon Processing Pathways and Activities

FIGS. 1 and 9 depict an embodiment of a biological pathway for makingadipic acid, using a sugar as the carbon source starting material. Anysuitable sugar can be used as the feedstock for the organism, (e.g.,6-carbon sugars (e.g., glucose, fructose), 5-carbon sugars (e.g.,xylose), the like or combinations thereof). The sugars are initiallymetabolized using naturally occurring and/or engineered pathways toyield malonyl CoA, which is depicted as the molecule entering the omegaoxidation pathway shown in FIG. 9. As depicted, the enzyme hexanoatesynthase converts two molecules of malonyl CoA and one molecule ofacetyl CoA to one molecule of hexanoic acid. In some embodiments acytochrome P450 enzyme converts hexanoic acid to 6-hydroxyhexanoic acid,which may be oxidized to 6-oxohexanoic acid via 6-hydroxyhexanoic aciddehydrogenase, or fatty alcohol oxidase. 6-oxohexanoic acid may beconverted to adipic acid by 6-oxohexanoic acid dehydrogenase.

A hexanoate synthase enzyme (HexS) is coded by hexonate synthase subunitalpha (HEXA) and hexanoate synthase subunit beta (HEXB) genes. In someembodiments, the HexS enzyme is endogenous to the host microorganism. Incertain embodiments, HEXA and HEXB genes may be isolated from a suitableorganism (e.g., Aspergillus parasiticus). In some embodiments, HEXA andHEXB orthologs, such as STCJ and STCK, also may be isolated fromsuitable organisms (e.g., Aspergillus nidulans).

Hexanoate is omega-hydroxylated by the enzyme cytochrome P450, therebygenerating a six carbon alcohol, in some embodiments. In certainembodiments, the cytochrome P450 enzyme is endogenous to the hostmicroorganism. In some embodiments the cytochrome P450 gene is isolatedfrom Bacillus megaterium and codes for a single subunit, soluble,cytoplasmic enzyme. Soluble or membrane bound cytochrome P450 fromcertain host organisms is specific for 6-carbon substrates and may beused in some embodiments.

Cytochrome P450 is reduced by the activity of cytochrome P450 reductase(CPR), thereby recycling cytochrome P450 to allow further enzymaticactivity. In certain embodiments, the CPR enzyme is endogenous to thehost microorganism. In some embodiments, host CPR activity can beincreased by increasing the number of copies of a CPR gene (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10 or more copies of the gene), by increasing theactivity of a promoter that regulates transcription of a CPR gene, or byincreasing the number of copies of a CPR gene and increasing theactivity of a promoter that regulates transcription of a CPR gene,thereby increasing the production of target product (e.g., adipic acid)via increased recycling of cytochrome P450. In certain embodiments, thepromoter can be a heterologous promoter (e.g., endogenous or exogenouspromoter). In some embodiments, the CPR gene is heterologous andexogenous and can be isolated from any suitable organism. Non-limitingexamples of organisms from which a CPR gene can be isolated include C.tropicalis, S. cerevisiae and Bacillus megaterium.

Oxidation of the alcohol to an aldehyde may be performed by an enzyme inthe fatty alcohol oxidase family (e.g., 6-hydroxyhexanoic aciddehydrogenase, omega hydroxyl fatty acid dehydrogenase), or an enzyme inthe aldehyde dehydrogenase family (e.g., 6-oxohexanoic aciddehydrogenase, omega oxo fatty acid dehydrogenase). The enzyme6-oxohexanoic acid dehydrogenase or omega oxo fatty acid dehydrogenasemay oxidize the aldehyde to the carboxylic acid adipic acid. In someembodiments, the enzymes 6-hydroxyhexanoic acid dehydrogenase, omegahydroxyl fatty acid dehydrogenase, fatty alcohol oxidase, 6-oxohexanoicacid dehydrogenase, or omega oxo fatty acid dehydrogenase, exist in ahost organism. Flux through these two steps may sometimes be augmentedby increasing the copy number of the enzymes, or by increasing theactivity of the promoter transcribing the genes. In some embodimentsalcohol and aldehyde dehydrogenases specific for six carbon substratesmay be isolated from another organism, for example Acinetobacter,Candida, Saccharomyces or Pseudomonas and inserted into the hostorganism.

FIG. 10 depicts an embodiment of a biological pathway for making adipicacid, using fats, oils, dicarboxylic acids, paraffins (e.g., linear,branched, substituted, saturated, unsaturated, the like and combinationsthereof), fatty alcohols, fatty acids, or the like, as the carbon sourcestarting material. Any suitable fatty alcohol, fatty acid, paraffin,dicarboxylic acid, fat or oil can be used as the feedstock for theorganism, (e.g., hexane, hexanoic acid, oleic acid, coconut oil, thelike or combinations thereof). Carbon sources with longer chain lengths(e.g., 8 carbons or greater in length) can be metabolized usingnaturally occurring and/or engineered pathways to yield molecules thatcan be further metabolized using the beta oxidation pathway shown inFIG. 10 and the lower portion of FIG. 11A. In some embodiments, theactivities in the pathway shown in FIG. 10 also can be engineered (e.g.,as described herein) to enhance metabolism and target product formation.The enzyme acyl-CoA ligase converts a long chain fatty alcohol, fattyacid or dicarboxylic acid and 1 molecule of acetyl-CoA into an acyl-CoAderivative of the long chain fatty alcohol, fatty acid or dicarboxylicacid with the conversion of ATP to AMP and inorganic phosphate, asdepicted in the first step of the reaction in FIG. 10. The term “betaoxidation pathway” as used herein, refers to a series of enzymaticactivities utilized to metabolize fatty alcohols, fatty acids, ordicarboxylic acids. The activities utilized to metabolize fattyalcohols, fatty acids, or dicarboxylic acids include, but are notlimited to, acyl-CoA ligase activity, acyl-CoA oxidase activity,enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activityand acetyl-CoA C-acyltransferase activity. The term “beta oxidationactivity” refers to any of the activities in the beta oxidation pathwayutilized to metabolize fatty alcohols, fatty acids or dicarboxylicacids. The term “omega oxidation activity” refers to any of theactivities in the omega oxidation pathway utilized to metabolize fattyalcohols, fatty acids, dicarboxylic acids, or sugars.

In certain embodiments, an Acyl-CoA ligase enzyme converts a long chainfatty alcohol, fatty acid or dicarboxylic acid into the acyl-CoAderivative, which may be oxidized to yield a trans-2,3-dehydroacyl-CoAderivative, by the activity of Acyl CoA oxidase (e.g., also known asacyl-CoA oxidoreductase and fatty acyl-coenzyme A oxidase). Thetrans-2,3-dehydroacyl-CoA derivative long chain fatty alcohol, fattyacid or dicarboxylic acid may be further converted to 3-hydroxyacyl-CoAby the activity of enoyl-CoA hydratase. 3-hydroxyacyl-CoA can beconverted to 3-oxoacyl-CoA by the activity of 3-hydroxyacyl-CoAdehydrogenase. 3-oxoacyl-CoA may be converted to an acyl-CoA molecule,shortened by 2 carbons and an acetyl-CoA, by the activity of Acetyl-CoAC-acyltransferase (e.g., also known as beta-ketothiolase andβ-ketothiolase). In some embodiments, acyl-CoA molecules may berepeatedly shortened by beta oxidation until a desired carbon chainlength is generated (e.g., 6 carbons, adipic acid). The shortened fattyacid can be further processed using omega oxidation to yield adipicacid.

An acyl-CoA ligase enzyme sometimes is encoded by the host organism andcan be added to generate an engineered organism. In some embodiments,host acyl-CoA ligase activity can be increased by increasing the numberof copies of an acyl-CoA ligase gene, by increasing the activity of apromoter that regulates transcription of an acyl-CoA ligase gene, or byincreasing the number copies of the gene and by increasing the activityof a promoter that regulates transcription of the gene, therebyincreasing production of target product (e.g., adipic acid) due toincreased carbon flux through the pathway. In certain embodiments, theacyl-CoA ligase gene can be isolated from any suitable organism.Non-limiting examples of organisms that include, or can be used asdonors for, acyl-CoA ligase enzymes include Candida, Saccharomyces, orYarrowia.

An enoyl-CoA hydratase enzyme catalyzes the addition of a hydroxyl groupand a proton to the unsaturated β-carbon on a fatty-acyl CoA andsometimes is encoded by the host organism and sometimes can be added togenerate an engineered organism. In certain embodiments, the enoyl-CoAhydratase activity is unchanged in a host or engineered organism. Insome embodiments, the host enoyl-CoA hydratase activity can be increasedby increasing the number of copies of an enoyl-CoA hydratase gene, byincreasing the activity of a promoter that regulates transcription of anenoyl-CoA hydratase gene, or by increasing the number copies of the geneand by increasing the activity of a promoter that regulatestranscription of the gene, thereby increasing the production of targetproduct (e.g., adipic acid) due to increased carbon flux through thepathway. In certain embodiments, the enoyl-CoA hydratase gene can beisolated from any suitable organism. Non-limiting examples of organismsthat include, or can be used as donors for, enoyl-CoA hydratase enzymesinclude Candida, Saccharomyces, or Yarrowia.

3-hydroxyacyl-CoA dehydrogenase enzyme catalyzes the formation of a3-ketoacyl-CoA by removal of a hydrogen from the newly formed hydroxylgroup created by the activity of enoyl-CoA hydratase. In someembodiments, the activity is encoded by the host organism and sometimescan be added or increased to generate an engineered organism. In certainembodiments, the 3-hydroxyacyl-CoA activity is unchanged in a host orengineered organism. In some embodiments, the host 3-hydroxyacyl-CoAdehydrogenase activity can be increased by increasing the number ofcopies of a 3-hydroxyacyl-CoA dehydrogenase gene, by increasing theactivity of a promoter that regulates transcription of a3-hydroxyacyl-CoA dehydrogenase gene, or by increasing the number copiesof the gene and by increasing the activity of a promoter that regulatestranscription of the gene, thereby increasing production of targetproduct (e.g., adipic acid) due to increased carbon flux through thepathway. In certain embodiments, the 3-hydroxyacyl-CoA dehydrogenasegene can be isolated from any suitable organism. Non-limiting examplesof organisms that include, or can be used as donors for,3-hydroxyacyl-CoA dehydrogenase enzymes include Candida, Saccharomyces,or Yarrowia.

An Acetyl-CoA C-acyltransferase (e.g., β-ketothiolase) enzyme catalyzesthe formation of a fatty acyl-CoA shortened by 2 carbons by cleavage ofthe 3-ketoacyl-CoA by the thiol group of another molecule of CoA. Thethiol is inserted between C-2 and C-3, which yields an acetyl CoAmolecule and an acyl CoA molecule that is two carbons shorter. AnAcetyl-CoA C-acyltransferase sometimes is encoded by the host organismand sometimes can be added to generate an engineered organism. Incertain embodiments, the acetyl-CoA C-acyltransferase activity isunchanged in a host or engineered organism. In some embodiments, thehost acetyl-CoA C-acyltransferase activity can be increased byincreasing the number of copies of an acetyl-CoA C-acyltransferase gene,or by increasing the activity of a promoter that regulates transcriptionof an acetyl-CoA C-acyltransferase gene, thereby increasing theproduction of target product (e.g., adipic acid) due to increased carbonflux through the pathway. In certain embodiments, the acetyl-CoAC-acyltransferase gene can be isolated from any suitable organism.Non-limiting examples of organisms that include, or can be used asdonors for, acetyl-CoA C-acyltransferase enzymes include Candida,Saccharomyces, or Yarrowia.

A microorganism may be modified and engineered to include or regulateone or more activities in an adipic acid pathway. The term “activity” asused herein refers to the functioning of a microorganism's natural orengineered biological pathways to yield various products includingadipic acid and its precursors. Adipic acid producing activity can beprovided by any non-mammalian source in certain embodiments. Suchsources include, without limitation, eukaryotes such as yeast and fungiand prokaryotes such as bacteria. In some embodiments, a reverseactivity in a pathway described herein can be altered (e.g., disrupted,reduced) to increase carbon flux through a beta oxidation pathway, anomega oxidation pathway, or a beta oxidation and omega oxidationpathway, towards the production of target product (e.g., adipic acid).In some embodiments, a genetic modification disrupts an activity in thebeta oxidation pathway, or disrupts a polynucleotide that encodes apolypeptide that carries out a forward reaction in the beta oxidationpathway, which renders beta oxidation activity undetectable. The term“undetectable” as used herein refers to an amount of an analyte that isbelow the limits of detection, using detection methods or assays known(e.g., described herein). In certain embodiments, the geneticmodification partially reduces beta oxidation activity. The term“partially reduces beta oxidation activity” as used here refers to alevel of activity in an engineered organism that is lower than the levelof activity found in the host or starting organism.

An activity within an engineered microorganism provided herein caninclude one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all) of thefollowing activities: 6-oxohexanoic acid dehydrogenase activity;6-hydroxyhexanoic acid dehydrogenase activity; hexanoate synthaseactivity; cytochrome P450 activity; cytochrome P450 reductase activity;fatty alcohol oxidase activity; acyl-CoA ligase activity, acyl-CoAoxidase activity; enoyl-CoA hydratase activity, 3-hydroxyacyl-CoAdehydrogenase activity, and thioesterase activity (e.g., acetyl-CoAC-acyltransferase, beta-ketothiolase). In certain embodiments, one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all) of the foregoingactivities is altered by way of a genetic modification. In someembodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all) ofthe foregoing activities is altered by way of (i) adding a heterologouspolynucleotide that encodes a polypeptide having the activity, and/or(ii) altering or adding a regulatory sequence that regulates theexpression of a polypeptide having the activity.

The term “6-oxohexanoic acid dehydrogenase activity” as used hereinrefers to conversion of 6-oxohexanoic acid to adipic acid. The6-oxohexanoic acid dehydrogenase activity can be provided by apolypeptide. In some embodiments, the polypeptide is encoded by aheterologous nucleotide sequence introduced to a host microorganism. Incertain embodiments, an endogenous polypeptide having the 6-oxohexanoicacid dehydrogenase activity is identified in the host microorganism, andthe host microorganism is genetically altered to increase the amount ofthe polypeptide produced (e.g., a heterologous promoter is introduced inoperable linkage with a polynucleotide that encodes the polypeptide; thecopy number of a polynucleotide that encodes the polypeptide isincreased (e.g., by introducing a plasmid that includes thepolynucleotide)). Nucleic acid sequences conferring 6-oxohexanoic aciddehydrogenase activity can be obtained from a number of sources,including Actinobacter, Norcardia, Pseudomonas and Xanthobacterbacteria. Examples of an amino acid sequence of a polypeptide having6-oxohexanoic acid dehydrogenase activity, and a nucleotide sequence ofa polynucleotide that encodes the polypeptide, are presented herein.Presence, absence or amount of 6-oxohexanoic acid dehydrogenase activitycan be detected by any suitable method known in the art. For an exampleof a detection method for alcohol oxidase or alcohol dehydrogenaseactivity (see Appl Environ Microbiol 70: 4872). In some embodiments,6-oxohexanoic acid dehydrogenase activity is not altered in a hostmicroorganism, and in certain embodiments, the activity is added orincreased in the engineered microorganism relative to the hostmicroorganism.

The term “omega oxo fatty acid dehydrogenase activity” as used hereinrefers to conversion of an omega oxo fatty acid to a dicarboxylic acid.The omega oxo fatty acid dehydrogenase activity can be provided by apolypeptide. In some embodiments, the polypeptide is encoded by aheterologous nucleotide sequence introduced to a host microorganism. Incertain embodiments, an endogenous polypeptide having the omega oxofatty acid dehydrogenase activity is identified in the hostmicroorganism, and the host microorganism is genetically altered toincrease the amount of the polypeptide produced (e.g., a heterologouspromoter is introduced in operable linkage with a polynucleotide thatencodes the polypeptide; the copy number of a polynucleotide thatencodes the polypeptide is increased (e.g., by introducing a plasmidthat includes the polynucleotide)). Nucleic acid sequences conferringomega oxo fatty acid dehydrogenase activity can be obtained from anumber of sources, including Actinobacter, Norcardia, Pseudomonas andXanthobacter bacteria. Examples of an amino acid sequence of apolypeptide having omega oxo fatty acid dehydrogenase activity and anucleotide sequence of a polynucleotide that encodes the polypeptide,are presented herein. Presence, absence or amount of omega oxo fattyacid dehydrogenase activity can be detected by any suitable method knownin the art. In some embodiments, omega oxo fatty acid dehydrogenaseactivity is not altered in a host microorganism, and in certainembodiments, the activity is added or increased in the engineeredmicroorganism relative to the host microorganism.

The term “6-hydroxyhexanoic acid dehydrogenase activity” as used hereinrefers to conversion of 6-hydroxyhexanoic acid to 6-oxohexanoic acid.The 6-hydroxyhexanoic acid dehydrogenase activity can be provided by apolypeptide. In some embodiments, the polypeptide is encoded by aheterologous nucleotide sequence introduced to a host microorganism. Incertain embodiments, an endogenous polypeptide having the6-hydroxyhexanoic acid dehydrogenase activity is identified in the hostmicroorganism, and the host microorganism is genetically altered toincrease the amount of the polypeptide produced (e.g., a heterologouspromoter is introduced in operable linkage with a polynucleotide thatencodes the polypeptide; the copy number of a polynucleotide thatencodes the polypeptide is increased (e.g., by introducing a plasmidthat includes the polynucleotide)). Nucleic acid sequences conferring6-hydroxyhexanoic acid dehydrogenase activity can be obtained from anumber of sources, including Actinobacter, Norcardia, Pseudomonas, andXanthobacter. Examples of an amino acid sequence of a polypeptide having6-hydroxyhexanoic acid dehydrogenase activity, and a nucleotide sequenceof a polynucleotide that encodes the polypeptide, are presented herein.Presence, absence or amount of 6-hydroxyhexanoic acid dehydrogenaseactivity can be detected by any suitable method known in the art. Anexample of such a method is described in Methods in Enzymology, 188:176. In some embodiments, 6-hydroxyhexanoic acid dehydrogenase activityis not altered in a host microorganism, and in certain embodiments, theactivity is added or increased in the engineered microorganism relativeto the host microorganism.

The term “omega hydroxyl fatty acid dehydrogenase activity” as usedherein refers to conversion of an omega hydroxyl fatty acid to an omegaoxo fatty acid. The omega hydroxyl fatty acid dehydrogenase activity canbe provided by a polypeptide. In some embodiments, the polypeptide isencoded by a heterologous nucleotide sequence introduced to a hostmicroorganism. In certain embodiments, an endogenous polypeptide havingthe omega hydroxyl fatty acid dehydrogenase activity is identified inthe host microorganism, and the host microorganism is geneticallyaltered to increase the amount of the polypeptide produced (e.g., aheterologous promoter is introduced in operable linkage with apolynucleotide that encodes the polypeptide; the copy number of apolynucleotide that encodes the polypeptide is increased (e.g., byintroducing a plasmid that includes the polynucleotide)). Nucleic acidsequences conferring omega hydroxyl fatty acid dehydrogenase activitycan be obtained from a number of sources, including Actinobacter,Norcardia, Pseudomonas and Xanthobacter bacteria. Examples of an aminoacid sequence of a polypeptide having omega hydroxyl fatty aciddehydrogenase activity and a nucleotide sequence of a polynucleotidethat encodes the polypeptide, are presented herein. Presence, absence oramount of omega hydroxyl fatty acid dehydrogenase activity can bedetected by any suitable method known in the art. In some embodiments,omega hydroxyl fatty acid dehydrogenase activity is not altered in ahost microorganism, and in certain embodiments, the activity is added orincreased in the engineered microorganism relative to the hostmicroorganism.

The term “hexanoate synthase activity” as used herein refers toconversion of acetyl-CoA and malonyl-CoA to hexanoic acid. Hexanoatesynthase activity may be provided by an enzyme that includes one or twosubunits (referred to hereafter as “subunit A” and/or “subunit B”). Thehexanoate synthase activity can be provided by a polypeptide. In someembodiments, the polypeptide is encoded by a heterologous nucleotidesequence introduced to a host microorganism. Nucleic acid sequencesconferring hexonate synthase activity can be obtained from a number ofsources, including Aspergillus parisiticus, for example. Examples of anamino acid sequence of a polypeptide having hexanoate synthase activity,and a nucleotide sequence of a polynucleotide that encodes thepolypeptide, are presented herein. In some embodiments, hexanoatesynthase activity is not altered in a host microorganism, and in certainembodiments, the activity is added or increased in the engineeredmicroorganism relative to the host microorganism.

Presence, absence or amount of hexanoate synthase activity can bedetected by any suitable method known in the art. An example of such amethod is described in Hexanoate synthase+thioesterase (Chemistry andBiology 9: 981-988). Briefly, an indicator strain may be prepared. Anindicator strain may be Bacillus subtilis containing a reporter gene(beta-galactosidase, green fluorescent protein, etc.) under control ofthe promoter regulated by LiaR, for example. An indicator strain alsomay be Candida tropicalis containing either the LiaR regulatablepromoter from Bacillus subtilis or the alkane inducible promoter for thenative gene for the peroxisomal 3-ketoacyl coenzyme A thiolase gene(CT-T3A), for example. Mutants with an improved functionality of HexS,thereby producing more hexanoic acid, can be plated onto a lawn ofindicator strain. Upon incubation and growth of both the test mutant andthe indicator strain, the appearance of a larger halo, which correlatesto the induction of the reporter strain compared to control strains,indicates a mutant with improved activity. In alternative approach,mutants are grown in conditions favoring production of hexanoyl CoA orhexanoic acid and lysed. Cell lysates are treated with proteases whichmay release hexanoic acid from the PKS. Clarified lysates may be spottedonto lawns of indicator strains to assess improved production. Inanother alternative approach, indicator strains are grown underconditions suitable to support expression of the reporter gene wheninduced by hexanoic acid. Dilutions of a known concentration of hexanoicacid are used to determine a standard curve. Lysates of the test straingrown under conditions favoring production of hexanoic acid are preparedand dilutions of the lysate added to the indicator strain. Indicatorstrains with lysates are placed under identical conditions as used todetermine the standard curve. The lysate dilutions that minimallysupport induction can be used to determine, quantitatively, the amountproduced when compared to the standard curve.

The term “monooxygenase activity” as used herein refers to inserting oneatom of oxygen from O₂ into an organic substrate (RH) and reducing theother oxygen atom to water. In some embodiments, monooxygenase activityrefers to incorporation of an oxygen atom onto a six-carbon organicsubstrate. In certain embodiments, monooxygenase activity refers toconversion of hexanoate to 6-hydroxyhexanoic acid. Monooxygenaseactivity can be provided by any suitable polypeptide, such as acytochrome P450 polypeptide (hereafter “CYP450”) in certain embodiments.Nucleic acid sequences conferring CYP450 activity can be obtained from anumber of sources, including Bacillus megaterium and may be induced inorganisms including but not limited to Candida tropicalis, Yarrowialipolytica, Aspergillus nidulans, and Aspergillus parasiticus. Examplesof oligonucleotide sequences utilized to isolate a polynucleotidesequence encoding a polypeptide having CYP450 activity (e.g., CYP52A15activity, CYP52A16 activity, or CYP52A15 activity and CYP52A16 activity)are presented herein. In some embodiments, monooxygenase activity is notaltered in a host microorganism, and in certain embodiments, theactivity is added or increased in the engineered microorganism relativeto the host microorganism.

Presence, absence or amount of cytochrome P450 activity can be detectedby any suitable method known in the art. For example, detection can beperformed by assaying a reaction containing cytochrome P450 (CYP52Afamily) and NADPH-cytochrome P450 reductase (see Appl Environ Microbiol69: 5983 and 5992). Briefly, cells are grown under standard conditionsand harvested for production of microsomes, which are used to detect CYPactivity. Microsomes are prepared by lysing cells in Tris-bufferedsucrose (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.25M sucrose). Differentialcentrifugation is performed first at 25,000×g then at 100,000×g topellet cell debris then microsomes, respectively. The microsome pelletis resuspended in 0.1M phosphate buffer (pH 7.5), 1 mM EDTA to a finalconcentration of approximately 10 mg protein/mL. A reaction mixturecontaining approximately 0.3 mg microsomes, 0.1 mM sodium hexanoate, 0.7mM NADPH, 50 mM Tris-HCl pH 7.5 in 1 mL is initiated by the addition ofNADPH and incubated at 37° C. for 10 minutes. The reaction is terminatedby addition of 0.25 mL 5M HCl and 0.25 mL 2.5 ug/mL 10-hydroxydecanoicacid is added as an internal standard (3.3 nmol). The mixture isextracted with 4.5 mL diethyl ether under NaCl-saturated conditions. Theorganic phase is transferred to a new tube and evaporated to dryness.The residue is dissolved in acetonitrile containing 10 mM3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1 mL of 15mg/mL 18-crown-6 in acetonitril saturated with K₂CO₃. The solution isincubated at 40° C. for 30 minutes before addition of 0.05 mL 2% aceticacid. The fluorescently labeled omega-hydroxy fatty acids are resolvedvia HPLC with detection at 430 nm and excitation at 355 nm (Yamada etal., 1991, AnalBiochem 199: 132-136). Optionally, specifically inducedCYP gene(s) may be detected by Northern blotting and/or quantitativeRT-PCR. (Craft et al., 2003, AppEnvironMicro 69: 5983-5991).

The term “monooxygenase reductase activity” as used herein refers to thetransfer of an electron from NAD(P)H, FMN, or FAD by way of an electrontransfer chain, reducing the ferric heme iron of cytochrome P450 to theferrous state. The term “monooxygenase reductase activity” as usedherein also can refer to the transfer of a second electron via theelectron transport system, reducing a dioxygen adduct to a negativelycharged peroxo group. In some embodiments, a monooxygenase activity candonate electrons from the two-electron donor NAD(P)H to the heme ofcytochrome P450 (e.g., monooxygenase activity) in a coupled two-stepreaction in which NAD(P)H can bind to the NAD(P)H-binding domain of thepolypeptide having the monooxygenase reductase activity and electronsare shuttled from NAD(P)H through FAD and FMN to the heme of themonooxygenase activity, thereby regenerating an active monooxygenaseactivity (e.g., cytochrome P450). Monooxygenase reductase activity canbe provided by any suitable polypeptide, such as a cytochrome P450reductase polypeptide (hereafter “CPR”) in certain embodiments. Nucleicacid sequences conferring CPR activity can be obtained from and/orinduced in a number of sources, including but not limited to Bacillusmegaterium, Candida tropicalis, Yarrowia lipolytica, Aspergillusnidulans, and Aspergillus parasiticus. Examples of oligonucleotidesequences utilized to isolate a polynucleotide sequence encoding apolypeptide having CPR activity are presented herein. In someembodiments, monooxygenase reductase activity is not altered in a hostmicroorganism, and in certain embodiments, the activity is added orincreased in the engineered microorganism relative to the hostmicroorganism.

Presence, absence or amount of CPR activity can be detected by anysuitable method known in the art. For example, an engineeredmicroorganism having an increased number of genes encoding a CPRactivity, relative to the host microorganism, could be detected usingquantitative nucleic acid detection methods (e.g., southern blotting,PCR, primer extension, the like and combinations thereof). An engineeredmicroorganism having increased expression of genes encoding a CPRactivity, relative to the host microorganism, could be detected usingquantitative expression based analysis (e.g., RT-PCR, western blotanalysis, northern blot analysis, the like and combinations thereof).Alternately, an enzymatic assay can be used to detect Cytochrome P450reductase activity, where the enzyme activity alters the opticalabsorbance at 550 nanometers of a substrate solution (Masters, B. S. S.,Williams, C. H., Kamin, H. (1967) Methods in Enzymology, X, 565-573).

The term “fatty alcohol oxidase activity” as used herein refers toinserting one atom of oxygen from O₂ into an organic substrate andreducing the other oxygen atom to peroxide. Fatty alcohol oxidaseactivity sometimes also is referred to as “long-chain-alcohol oxidaseactivity”, “long-chain-alcohol:oxygen oxidoreductase activity”, “fattyalcohol:oxygen oxidoreductase activity” and “long-chain fatty acidoxidase activity”. In some embodiments, fatty alcohol oxidase activityrefers to incorporation of an oxygen atom onto a six-carbon organicsubstrate. In certain embodiments, fatty alcohol oxidase activity refersto the conversion of 6-hydroxyhexanoic acid into 6-oxohexanoic acid. Insome embodiments, fatty alcohol oxidase activity refers to theconversion of an omega hydroxyl fatty acid into an omega oxo fatty acid.A Fatty alcohol oxidase (FAO) activity can be provided by any suitablepolypeptide, such as a fatty alcohol oxidase peptide, along-chain-alcohol oxidase peptide, a long-chain-alcohol:oxygenoxidoreductase peptide, a fatty alcohol:oxygen oxidoreductase peptideand a long-chain fatty acid oxidase peptide. Nucleic acid sequencesconferring FAO activity can be obtained from a number of sources,including but not limited to Candida tropicalis, Candida cloacae,Yarrowia lipolytica, and Arabidopsis thaliana. Examples of amino acidsequences of polypeptides having FAO activity, and nucleotide sequencesof polynucleotides that encode the polypeptides, are presented herein.In some embodiments, fatty alcohol oxidase activity is not altered in ahost microorganism, and in certain embodiments, the activity is added orincreased in the engineered microorganism relative to the hostmicroorganism.

Presence, absence or amount of FAO activity can be detected by anysuitable method known in the art. For example, an engineeredmicroorganism having an increased number of genes encoding an FAOactivity, relative to the host microorganism, could be detected usingquantitative nucleic acid detection methods (e.g., southern blotting,PCR, primer extension, the like and combinations thereof). An engineeredmicroorganism having increased expression of genes encoding an FAOactivity, relative to the host microorganism, could be detected usingquantitative expression based analysis (e.g., RT-PCR, western blotanalysis, northern blot analysis, the like and combinations thereof).Alternately, an enzymatic assay can be used to detect fatty alcoholoxidase activity as described in Eirich et al, 2004, or as modified inthe Examples herein.

The term “acyl-CoA oxidase activity” as used herein refers to theoxidation of a long chain fatty-acyl-CoA to a trans-2,3-dehydroacyl-CoAfatty alcohol. In some embodiments, the acyl-CoA activity is from aperoxisome. In certain embodiments, the acyl-CoA oxidase activity is aperoxisomal acyl-CoA oxidase (POX) activity, carried out by a POXpolypeptide. In some embodiments the acyl-CoA oxidase activity isencoded by the host organism and sometimes can be altered to generate anengineered organism. Acyl-CoA oxidase activity is encoded by the POX4and POX5 genes of C. tropicalis. In certain embodiments, endogenousacyl-CoA oxidase activity can be increased. In some embodiments,acyl-CoA oxidase activity of the POX4 polypeptide or the POX5polypeptide can be altered independently of each other (e.g., increaseactivity of POX4 alone, POX5 alone, increase one and disrupt the other,and the like). Increasing the activity of one POX activity, whiledisrupting the activity of another POX activity, may alter the specificactivity of acyl-CoA oxidase with respect to carbon chain length, whilemaintaining or increasing overall flux through the beta oxidationpathway, in certain embodiments.

FIGS. 15A-15C graphically illustrate the units of acyl-CoA oxidaseactivity expressed as units (U) per milligram of protein (Y axis) invarious strains of Candida tropicalis induced by feedstocks of specificchain length (Picataggio et al. 1991 Molecular and Cellular Biology 11:4333-4339). Isolated protein was assayed for acyl-CoA oxidase activityusing carbon chains of various length (X axis). The X and Y axes inFIGS. 15A-15C represent substantially similar data. FIG. 15A illustratesacyl-CoA oxidase activity as measured in a strain having a fullcomplement of POX genes (e.g., POX4 and POX5 are active). FIG. 15Billustrates acyl-CoA oxidase activity as measured in a strain having adisrupted POX5 gene. The activity encoded by the functional POX4 geneexhibits a higher specific activity for acyl-CoA molecules with shortercarbon chain lengths (e.g., less than 10 carbons). The results of thePOX5 disrupted strain also are presented numerically in the table inFIG. 15B. FIG. 15C illustrates acyl-CoA oxidase activity as measured ina strain having a disrupted POX4 gene. The activity encoded by thefunctional POX5 gene exhibits a narrow peak of high specific activityfor acyl-CoA molecules 12 carbons in length, with a lower specificactivity for molecules 10 carbons in length. The results of the POX4disrupted strain are presented numerically in the table in FIG. 15C.

In certain embodiments, host acyl-CoA oxidase activity of one of the POXgenes can be increased by genetically altering (e.g., increasing) theamount of the polypeptide produced (e.g., a strongly transcribed orconstitutively expressed heterologous promoter is introduced in operablelinkage with a polynucleotide that encodes the polypeptide; the copynumber of a polynucleotide that encodes the polypeptide is increased(e.g., by introducing a plasmid that includes the polynucleotide,integration of additional copies in the host genome)). In someembodiments, the host acyl-CoA oxidase activity can be decreased bydisruption (e.g., knockout, insertion mutagenesis, the like andcombinations thereof) of an acyl-CoA oxidase gene, or by decreasing theactivity of the promoter (e.g., addition of repressor sequences to thepromoter or 5′UTR) which transcribes an acyl-CoA oxidase gene.

A noted above, disruption of nucleotide sequences encoding POX4, POX 5,or POX4 and POX5 sometimes can alter pathway efficiency, specificityand/or specific activity with respect to metabolism of carbon chains ofdifferent lengths (e.g., carbon chains including fatty alcohols, fattyacids, paraffins, dicarboxylic acids of between about 1 and about 60carbons in length). In some embodiments, the nucleotide sequence ofPOX4, POX5, or POX4 and POX5 is disrupted with a URA3 nucleotidesequence encoding a selectable marker, and introduced to a hostmicroorganism, thereby generating an engineered organism deficient inPOX4, POX5 or POX4 and POX5 activity. Nucleic acid sequences encodingPOX4 and POX5 can be obtained from a number of sources, includingCandida tropicalis, for example. Examples of POX4 and POX5 amino acidsequences and nucleotide sequences of polynucleotides that encode thepolypeptides, are presented herein.

Presence, absence or amount of POX4 and/or POX5 activity can be detectedby any suitable method known in the art. For example, using enzymaticassays as described in Shimizu et al, 1979, and as described herein inthe Examples. Alternatively, nucleic acid sequences representing nativeand/or disrupted POX4 and POX5 sequences also can be detected usingnucleic acid detection methods (e.g., PCR, primer extension, nucleicacid hybridization, the like and combinations thereof), or quantitativeexpression based analysis (e.g., RT-PCR, western blot analysis, northernblot analysis, the like and combinations thereof), where the engineeredorganism exhibits decreased RNA and/or polypeptide levels as compared tothe host organism.

The term “thioesterase activity” as used herein refers to removal ofCoenzyme A from hexanoate. The thioesterase activity can be provided bya polypeptide. In some embodiments, the polypeptide is encoded by aheterologous nucleotide sequence introduced to a host microorganism.Nucleic acid sequences conferring thioesterase activity can be obtainedfrom a number of sources, including Cuphea lanceolata. Examples of suchpolypeptides include, without limitation, acyl-(ACP) thioesterase type Bfrom Cuphea lanceolata, encoded by the nucleotide sequences referencedby accession number CAB60830 at the World Wide Web Uniform ResourceLocator (URL) ncbi.nlm.nih.gov of the National Center for BiotechnologyInformation (NCBI).

Presence, absence or amount of thioesterase activity can be detected byany suitable method known in the art. An example of such a method isdescribed Chemistry and Biology 9: 981-988. In some embodiments,thioesterase activity is not altered in a host microorganism, and incertain embodiments, the activity is added or increased in theengineered microorganism relative to the host microorganism. In someembodiments, a polypeptide having thioesterase activity is linked toanother polypeptide (e.g., a hexanoate synthase A or hexanoate synthaseB polypeptide). A non-limiting example of an amino acid sequence (oneletter code sequence) for a polypeptide having thioesterase activity isprovided hereafter:

MVAAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPNAGFQVKANASAHPKANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAITTVFVAAEKQWTMLDRKSKRPDMLVDSVGLKSIVRDGLVSRQSFLIRSYEIGADRTASIETLMNHLQETSINHCKSLGLLNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLHKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSAS

The term “a genetic modification that results in substantial hexanoateusage by monooxygenase activity” as used herein refers to a geneticalteration of a host microorganism that reduces an endogenous activitythat converts hexanoate to another product. In some embodiments, anendogenous activity that converts hexanoate to a toxin (e.g., in fungus)is reduced. In certain embodiments, a polyketide synthase activity isreduced. Such alterations can advantageously increase yields of endproducts, such as adipic acid.

The term “polyketide synthase activity” as used herein refers to thealteration of hexanoic acid by the polyketide synthase enzyme (PKS) as astep in the production of other products including mycotoxin. The PKSactivity can be provided by a polypeptide. Examples of such polypeptidesinclude, without limitation, an Aspergillus parasiticus enzymereferenced by accession number AAS66004 at the World Wide Web UniformResource Locator (URL) ncbi.nlm.nih.gov of the National Center forBiotechnology Information (NCBI). In certain embodiments, a PKS enzymeuses hexanoic acid generated by hexanoate synthase as a substrate and acomponent of the Aspergillus NorS multienzyme complex, a closelyassociated gene cluster involved in the synthesis of various productsincluding mytoxin. Accordingly, a PKS activity sometimes is altered tofree hexanoic acid for an engineered adipic acid pathway. In someembodiments PKS activity is diminished or blocked. In certainembodiments the PKS enzyme is engineered to substitute thioesteraseactivity for PKS activity. Presence, absence, or amount of PKS activitycan be detected by any suitable method known in the art, such as thatdescribed in Watanabe C and Townsend C (2002) Initial characterizationof a type I fatty acid synthase and polyketide synthase multienzymecomplex NorS in the biosynthesis of aflatoxin B1. Chemistry and Biology9: 981-988.

A non-limiting example of an amino acid sequence (one letter codesequence) of a polypeptide having polyketide synthase activity isprovided hereafter:

MAQSRQLFLFGDQTADFVPKLRSLLSVQDSPILAAFLDQSHYVVRAQMLQSMNTVDHKLARTADLRQMVQKYVDGKLTPAFRTALVCLCQLGCFIREYEESGNMYPQPSDSYVLGFCMGSLAAVAVSCSRSLSELLPIAVQTVLIAFRLGLCALEMRDRVDGCSDDRGDPWSTIVWGLDPQQARDQIEVFCRTTNVPQTRRPWISCISKNAITLSGSPSTLRAFCAMPQMAQHRTAPIPICLPAHNGALFTQADITTILDTTPTTPWEQLPGQIPYISHVTGNVVQTSNYRDLIEVALSETLLEQVRLDLVETGLPRLLQSRQVKSVTIVPFLTRMNETMSNILPDSFISTETRTDTGRAIPASGRPGAGKCKLAIVSMSGRFPESPTTESFWDLLYKGLDVCKEVPRRRWDINTHVDPSGKARNKGATKWGCWLDFSGDFDPRFFGISPKEAPQMDPAQRMALMSTYEAMERAGLVPDTTPSTQRDRIGVFHGVTSNDWMETNTAQNIDTYFITGGNRGFIPGRINFCFEFAGPSYTNDTACSSSLAAIHLACNSLWRGDCDTAVAGGTNMIYTPDGHTGLDKGFFLSRTGNCKPYDDKADGYCRAEGVGTVFIKRLEDALADNDPILGVILDAKTNHSAMSESMTRPHVGAQIDNMTAALNTTGLHPNDFSYIEMHGTGTQVGDAVEMESVLSVFAPSETARKADQPLFVGSAKANVGHGEGVSGVTSLIKVLMMMQHDTIPPHCGIKPGSKINRNFPDLGARNVHIAFEPKPWPRTHTPRRVLINNFSAAGGNTALIVEDAPERHWPTEKDPRSSHIVALSAHVGASMKTNLERLHQYLLKNPHTDLAQLSYTTTARRWHYLHRVSVTGASVEEVTRKLEMAIQNGDGVSRPKSKPKILFAFTGQGSQYATMGKQVYDAYPSFREDLEKFDRLAQSHGFPSFLHVCTSPKGDVEEMAPVVVQLAITCLQMALTNLMTSFGIRPDVTVGHSLGEFAALYAAGVLSASDVVYLVGQRAELLQERCQRGTHAMLAVKATPEALSQWIQDHDCEVACINGPEDTVLSGTTKNVAEVQRAMTDNGIKCTLLKLPFAFHSAQVQPILDDFEALAQGATFAKPQLLILSPLLRTEIHEQGVVTPSYVAQHCRHTVDMAQALRSAREKGLIDDKTLVIELGPKPLISGMVKMTLGDKISTLPTLAPNKAIWPSLQKILTSVYTGGWDINWKKYHAPFASSQKVVDLPSYGWDLKDYYIPYQGDWCLHRHQQDCKCAAPGHEIKTADYQVPPESTPHRPSKLDPSKEAFPEIKTTTTLHRVVEETTKPLGATLVVETDISRKDVNGLARGHLVDGIPLCTPSFYADIAMQVGQYSMQRLRAGHPGAGAIDGLVDVSDMVVDKALVPHGKGPQLLRTTLTMEWPPKAAATTRSAKVKFATYFADGKLDTEHASCTVRFTSDAQLKSLRRSVSEYKTHIRQLHDGHAKGQFMRYNRKTGYKLMSSMARFNPDYMLLDYLVLNEAENEAASGVDFSLGSSEGTFAAHPAHVDAITQVAGFAMNANDNVDIEKQVYVNHGWDSFQIYQPLDNSKSYQVYTKMGQAKENDLVHGDVVVLDGEQIVAFFRGLTLRSVPRGALRVVLQTTVKKADRQLGFKTMPSPPPPTTTMPISPYKPANTQVSSQAIPAEATHSHTPPQPKHSPVPETAGSAPAAKGVGVSNEKLDAVMRVVSEESGIALEELTDDSNFADMGIDSLSSMVIGSRFREDLGLDLGPEFSLFIDCTTVRALKDFMLGSGDAGSGSNVEDPPPSATPGINPETDWSSSASDSIFASEDHGHSSESGADTGSPPALDLKPYCRPSTSVVLQGLPMVARKTLFMLPDGGGSAFSYASLPRLKSDTAVVGLNCPYARDPENMNCTHGAMIESFCNEIRRRQPRGPYHLGGWSSGGAFAYVVAEALVNQGEEVHSLIIIDAPIPQAMEQLPRAFYEHCNSIGLFATQPGASPDGSTEPPSYLIPHFTAVVDVMLDYKLAPLHARRMPKVGIVWAADTVMDERDAPKMKGMHFMIQKRTEFGPDGWDTIMPGASFDIVRADGANHFTLMQKEHVSIISDLIDRVMA

The terms “a genetic modification that reduces 6-hydroxyhexanoic acidconversion” or “a genetic modification that reduces omega hydroxyl fattyacid conversion” as used herein refer to genetic alterations of a hostmicroorganism that reduce an endogenous activity that converts6-hydroxyhexanoic acid to another product. In some embodiments, anendogenous 6-hydroxyhexanoic acid dehydrogenase activity is reduced.Such alterations can advantageously increase the amount of6-hydroxyhexanoic acid, which can be purified and further processed.

The term “a genetic modification that reduces beta-oxidation activity”as used herein refers to a genetic alteration of a host microorganismthat reduces an endogenous activity that oxidizes a beta carbon ofcarboxylic acid containing organic molecules. In certain embodiments,the organic molecule is a six carbon molecule, and sometimes containsone or two carboxylic acid moieties located at a terminus of themolecule (e.g., adipic acid). Such alterations can advantageouslyincrease yields of end products, such as adipic acid.

Polynucleotides and Polypeptides

A nucleic acid (e.g., also referred to herein as nucleic acid reagent,target nucleic acid, target nucleotide sequence, nucleic acid sequenceof interest or nucleic acid region of interest) can be from any sourceor composition, such as DNA, cDNA, gDNA (genomic DNA), RNA, siRNA (shortinhibitory RNA), RNAi, tRNA or mRNA, for example, and can be in any form(e.g., linear, circular, supercoiled, single-stranded, double-stranded,and the like). A nucleic acid can also comprise DNA or RNA analogs(e.g., containing base analogs, sugar analogs and/or a non-nativebackbone and the like). It is understood that the term “nucleic acid”does not refer to or infer a specific length of the polynucleotidechain, thus polynucleotides and oligonucleotides are also included inthe definition. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracilbase is uridine.

A nucleic acid sometimes is a plasmid, phage, autonomously replicatingsequence (ARS), centromere, artificial chromosome, yeast artificialchromosome (e.g., YAC) or other nucleic acid able to replicate or bereplicated in a host cell. In certain embodiments a nucleic acid can befrom a library or can be obtained from enzymatically digested, shearedor sonicated genomic DNA (e.g., fragmented) from an organism ofinterest. In some embodiments, nucleic acid subjected to fragmentationor cleavage may have a nominal, average or mean length of about 5 toabout 10,000 base pairs, about 100 to about 1,000 base pairs, about 100to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000base pairs. Fragments can be generated by any suitable method in theart, and the average, mean or nominal length of nucleic acid fragmentscan be controlled by selecting an appropriate fragment-generatingprocedure by the person of ordinary skill. In some embodiments, thefragmented DNA can be size selected to obtain nucleic acid fragments ofa particular size range.

Nucleic acid can be fragmented by various methods known to the person ofordinary skill, which include without limitation, physical, chemical andenzymic processes. Examples of such processes are described in U.S.Patent Application Publication No. 20050112590 (published on May 26,2005, entitled “Fragmentation-based methods and systems for sequencevariation detection and discovery,” naming Van Den Boom et al.). Certainprocesses can be selected by the person of ordinary skill to generatenon-specifically cleaved fragments or specifically cleaved fragments.Examples of processes that can generate non-specifically cleavedfragment sample nucleic acid include, without limitation, contactingsample nucleic acid with apparatus that expose nucleic acid to shearingforce (e.g., passing nucleic acid through a syringe needle; use of aFrench press); exposing sample nucleic acid to irradiation (e.g., gamma,x-ray, UV irradiation; fragment sizes can be controlled by irradiationintensity); boiling nucleic acid in water (e.g., yields about 500 basepair fragments) and exposing nucleic acid to an acid and base hydrolysisprocess.

Nucleic acid may be specifically cleaved by contacting the nucleic acidwith one or more specific cleavage agents. The term “specific cleavageagent” as used herein refers to an agent, sometimes a chemical or anenzyme that can cleave a nucleic acid at one or more specific sites.Specific cleavage agents often will cleave specifically according to aparticular nucleotide sequence at a particular site. Examples of enzymicspecific cleavage agents include without limitation endonucleases (e.g.,DNase (e.g., DNase I, II); RNase (e.g., RNase E, F, H, P); Cleavase™enzyme; Taq DNA polymerase; E. coli DNA polymerase I and eukaryoticstructure-specific endonucleases; murine FEN-1 endonucleases; type I, IIor III restriction endonucleases such as Acc I, Afl III, Alu I, Alw44 I,Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl I. Bgl II, BlnI, Bsm I, BssH II, BstE II, Cfo I, Cla I, Dde I, Dpn I, Dra I, EclX I,EcoR I, EcoR I, EcoR II, EcoR V, Hae II, Hae II, Hind III, Hind III, HpaI, Hpa II, Kpn I, Ksp I, Mlu I, MluN I, Msp I, Nci I, Nco I, Nde I, NdeII, Nhe I, Not I, Nru I, Nsi I, Pst I, Pvu I, Pvu II, Rsa I, Sac I, SalI, Sau3A I, Sca I, ScrF I, Sfi I, Sma I, Spe I, Sph I, Ssp I, Stu I, StyI, Swa I, Taq I, Xba I, Xho I); glycosylases (e.g., uracil-DNAglycosylase (UDG), 3-methyladenine DNA glycosylase, 3-methyladenine DNAglycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNAglycosylase, thymine mismatch-DNA glycosylase, hypoxanthine-DNAglycosylase, 5-Hydroxymethyluracil DNA glycosylase (HmUDG),5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNAglycosylase); exonucleases (e.g., exonuclease III); ribozymes, andDNAzymes. Sample nucleic acid may be treated with a chemical agent, orsynthesized using modified nucleotides, and the modified nucleic acidmay be cleaved. In non-limiting examples, sample nucleic acid may betreated with (i) alkylating agents such as methylnitrosourea thatgenerate several alkylated bases, including N3-methyladenine andN3-methylguanine, which are recognized and cleaved by alkyl purineDNA-glycosylase; (ii) sodium bisulfite, which causes deamination ofcytosine residues in DNA to form uracil residues that can be cleaved byuracil N-glycosylase; and (iii) a chemical agent that converts guanineto its oxidized form, 8-hydroxyguanine, which can be cleaved byformamidopyrimidine DNA N-glycosylase. Examples of chemical cleavageprocesses include without limitation alkylation, (e.g., alkylation ofphosphorothioate-modified nucleic acid); cleavage of acid lability ofP3′-N5′-phosphoroamidate-containing nucleic acid; and osmium tetroxideand piperidine treatment of nucleic acid.

As used herein, the term “complementary cleavage reactions” refers tocleavage reactions that are carried out on the same nucleic acid usingdifferent cleavage reagents or by altering the cleavage specificity ofthe same cleavage reagent such that alternate cleavage patterns of thesame target or reference nucleic acid or protein are generated. Incertain embodiments, nucleic acids of interest may be treated with oneor more specific cleavage agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore specific cleavage agents) in one or more reaction vessels (e.g.,nucleic acid of interest is treated with each specific cleavage agent ina separate vessel).

A nucleic acid suitable for use in the embodiments described hereinsometimes is amplified by any amplification process known in the art(e.g., PCR, RT-PCR and the like). Nucleic acid amplification may beparticularly beneficial when using organisms that are typicallydifficult to culture (e.g., slow growing, require specialize cultureconditions and the like). The terms “amplify”, “amplification”,“amplification reaction”, or “amplifying” as used herein refer to any invitro processes for multiplying the copies of a target sequence ofnucleic acid. Amplification sometimes refers to an “exponential”increase in target nucleic acid. However, “amplifying” as used hereincan also refer to linear increases in the numbers of a select targetsequence of nucleic acid, but is different than a one-time, singleprimer extension step. In some embodiments, a limited amplificationreaction, also known as pre-amplification, can be performed.Pre-amplification is a method in which a limited amount of amplificationoccurs due to a small number of cycles, for example 10 cycles, beingperformed. Pre-amplification can allow some amplification, but stopsamplification prior to the exponential phase, and typically producesabout 500 copies of the desired nucleotide sequence(s). Use ofpre-amplification may also limit inaccuracies associated with depletedreactants in standard PCR reactions.

In some embodiments, a nucleic acid reagent sometimes is stablyintegrated into the chromosome of the host organism, or a nucleic acidreagent can be a deletion of a portion of the host chromosome, incertain embodiments (e.g., genetically modified organisms, wherealteration of the host genome confers the ability to selectively orpreferentially maintain the desired organism carrying the geneticmodification). Such nucleic acid reagents (e.g., nucleic acids orgenetically modified organisms whose altered genome confers a selectabletrait to the organism) can be selected for their ability to guideproduction of a desired protein or nucleic acid molecule. When desired,the nucleic acid reagent can be altered such that codons encode for (i)the same amino acid, using a different tRNA than that specified in thenative sequence, or (ii) a different amino acid than is normal,including unconventional or unnatural amino acids (including detectablylabeled amino acids). As described herein, the term “native sequence”refers to an unmodified nucleotide sequence as found in its naturalsetting (e.g., a nucleotide sequence as found in an organism).

A nucleic acid or nucleic acid reagent can comprise certain elementsoften selected according to the intended use of the nucleic acid. Any ofthe following elements can be included in or excluded from a nucleicacid reagent. A nucleic acid reagent, for example, may include one ormore or all of the following nucleotide elements: one or more promoterelements, one or more 5′ untranslated regions (5′UTRs), one or moreregions into which a target nucleotide sequence may be inserted (an“insertion element”), one or more target nucleotide sequences, one ormore 3′ untranslated regions (3′UTRs), and one or more selectionelements. A nucleic acid reagent can be provided with one or more ofsuch elements and other elements may be inserted into the nucleic acidbefore the nucleic acid is introduced into the desired organism. In someembodiments, a provided nucleic acid reagent comprises a promoter,5′UTR, optional 3′UTR and insertion element(s) by which a targetnucleotide sequence is inserted (i.e., cloned) into the nucleotide acidreagent. In certain embodiments, a provided nucleic acid reagentcomprises a promoter, insertion element(s) and optional 3′UTR, and a 5′UTR/target nucleotide sequence is inserted with an optional 3′UTR. Theelements can be arranged in any order suitable for expression in thechosen expression system (e.g., expression in a chosen organism, orexpression in a cell free system, for example), and in some embodimentsa nucleic acid reagent comprises the following elements in the 5′ to 3′direction: (1) promoter element, 5′UTR, and insertion element(s); (2)promoter element, 5′UTR, and target nucleotide sequence; (3) promoterelement, 5′UTR, insertion element(s) and 3′UTR; and (4) promoterelement, 5′UTR, target nucleotide sequence and 3′UTR.

A promoter element typically is required for DNA synthesis and/or RNAsynthesis. A promoter element often comprises a region of DNA that canfacilitate the transcription of a particular gene, by providing a startsite for the synthesis of RNA corresponding to a gene. Promotersgenerally are located near the genes they regulate, are located upstreamof the gene (e.g., 5′ of the gene), and are on the same strand of DNA asthe sense strand of the gene, in some embodiments. In some embodiments,a promoter element can be isolated from a gene or organism and insertedin functional connection with a polynucleotide sequence to allow alteredand/or regulated expression. A non-native promoter (e.g., promoter notnormally associated with a given nucleic acid sequence) used forexpression of a nucleic acid often is referred to as a heterologouspromoter. In certain embodiments, a heterologous promoter and/or a 5′UTRcan be inserted in functional connection with a polynucleotide thatencodes a polypeptide having a desired activity as described herein. Theterms “operably linked” and “in functional connection with” as usedherein with respect to promoters, refer to a relationship between acoding sequence and a promoter element. The promoter is operably linkedor in functional connection with the coding sequence when expressionfrom the coding sequence via transcription is regulated, or controlledby, the promoter element. The terms “operably linked” and “in functionalconnection with” are utilized interchangeably herein with respect topromoter elements.

A promoter often interacts with a RNA polymerase. A polymerase is anenzyme that catalyses synthesis of nucleic acids using a preexistingnucleic acid reagent. When the template is a DNA template, an RNAmolecule is transcribed before protein is synthesized. Enzymes havingpolymerase activity suitable for use in the present methods include anypolymerase that is active in the chosen system with the chosen templateto synthesize protein. In some embodiments, a promoter (e.g., aheterologous promoter) also referred to herein as a promoter element,can be operably linked to a nucleotide sequence or an open reading frame(ORF). Transcription from the promoter element can catalyze thesynthesis of an RNA corresponding to the nucleotide sequence or ORFsequence operably linked to the promoter, which in turn leads tosynthesis of a desired peptide, polypeptide or protein.

Promoter elements sometimes exhibit responsiveness to regulatorycontrol. Promoter elements also sometimes can be regulated by aselective agent. That is, transcription from promoter elements sometimescan be turned on, turned off, up-regulated or down-regulated, inresponse to a change in environmental, nutritional or internalconditions or signals (e.g., heat inducible promoters, light regulatedpromoters, feedback regulated promoters, hormone influenced promoters,tissue specific promoters, oxygen and pH influenced promoters, promotersthat are responsive to selective agents (e.g., kanamycin) and the like,for example). Promoters influenced by environmental, nutritional orinternal signals frequently are influenced by a signal (direct orindirect) that binds at or near the promoter and increases or decreasesexpression of the target sequence under certain conditions.

Non-limiting examples of selective or regulatory agents that caninfluence transcription from a promoter element used in embodimentsdescribed herein include, without limitation, (1) nucleic acid segmentsthat encode products that provide resistance against otherwise toxiccompounds (e.g., antibiotics); (2) nucleic acid segments that encodeproducts that are otherwise lacking in the recipient cell (e.g.,essential products, tRNA genes, auxotrophic markers); (3) nucleic acidsegments that encode products that suppress the activity of a geneproduct; (4) nucleic acid segments that encode products that can bereadily identified (e.g., phenotypic markers such as antibiotics (e.g.,β-lactamase), β-galactosidase, green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), cyanfluorescent protein (CFP), and cell surface proteins); (5) nucleic acidsegments that bind products that are otherwise detrimental to cellsurvival and/or function; (6) nucleic acid segments that otherwiseinhibit the activity of any of the nucleic acid segments described inNos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acidsegments that bind products that modify a substrate (e.g., restrictionendonucleases); (8) nucleic acid segments that can be used to isolate oridentify a desired molecule (e.g., specific protein binding sites); (9)nucleic acid segments that encode a specific nucleotide sequence thatcan be otherwise non-functional (e.g., for PCR amplification ofsubpopulations of molecules); (10) nucleic acid segments that, whenabsent, directly or indirectly confer resistance or sensitivity toparticular compounds; (11) nucleic acid segments that encode productsthat either are toxic or convert a relatively non-toxic compound to atoxic compound (e.g., Herpes simplex thymidine kinase, cytosinedeaminase) in recipient cells; (12) nucleic acid segments that inhibitreplication, partition or heritability of nucleic acid molecules thatcontain them; and/or (13) nucleic acid segments that encode conditionalreplication functions, e.g., replication in certain hosts or host cellstrains or under certain environmental conditions (e.g., temperature,nutritional conditions, and the like). In some embodiments, theregulatory or selective agent can be added to change the existing growthconditions to which the organism is subjected (e.g., growth in liquidculture, growth in a fermentor, growth on solid nutrient plates and thelike for example).

In some embodiments, regulation of a promoter element can be used toalter (e.g., increase, add, decrease or substantially eliminate) theactivity of a peptide, polypeptide or protein (e.g., enzyme activity forexample). For example, a microorganism can be engineered by geneticmodification to express a nucleic acid reagent that can add a novelactivity (e.g., an activity not normally found in the host organism) orincrease the expression of an existing activity by increasingtranscription from a homologous or heterologous promoter operably linkedto a nucleotide sequence of interest (e.g., homologous or heterologousnucleotide sequence of interest), in certain embodiments. In someembodiments, a microorganism can be engineered by genetic modificationto express a nucleic acid reagent that can decrease expression of anactivity by decreasing or substantially eliminating transcription from ahomologous or heterologous promoter operably linked to a nucleotidesequence of interest, in certain embodiments.

In some embodiments the activity can be altered using recombinant DNAand genetic techniques known to the artisan. Methods for engineeringmicroorganisms are further described herein. Tables herein providenon-limiting lists of yeast promoters that are up-regulated by oxygen,yeast promoters that are down-regulated by oxygen, yeast transcriptionalrepressors and their associated genes, DNA binding motifs as determinedusing the MEME sequence analysis software. Potential regulator bindingmotifs can be identified using the program MEME to search intergenicregions bound by regulators for overrepresented sequences. For eachregulator, the sequences of intergenic regions bound with p-values lessthan 0.001 were extracted to use as input for motif discovery. The MEMEsoftware was run using the following settings: a motif width rangingfrom 6 to 18 bases, the “zoops” distribution model, a 6^(th) orderMarkov background model and a discovery limit of 20 motifs. Thediscovered sequence motifs were scored for significance by two criteria:an E-value calculated by MEME and a specificity score. The motif withthe best score using each metric is shown for each regulator. All motifspresented are derived from datasets generated in rich growth conditionswith the exception of a previously published dataset for epitope-taggedGal4 grown in galactose.

In some embodiments, the altered activity can be found by screening theorganism under conditions that select for the desired change inactivity. For example, certain microorganisms can be adapted to increaseor decrease an activity by selecting or screening the organism inquestion on a media containing substances that are poorly metabolized oreven toxic. An increase in the ability of an organism to grow asubstance that is normally poorly metabolized would result in anincrease in the growth rate on that substance, for example. A decreasein the sensitivity to a toxic substance might be manifested by growth onhigher concentrations of the toxic substance, for example. Geneticmodifications that are identified in this manner sometimes are referredto as naturally occurring mutations or the organisms that carry them cansometimes be referred to as naturally occurring mutants. Modificationsobtained in this manner are not limited to alterations in promotersequences. That is, screening microorganisms by selective pressure, asdescribed above, can yield genetic alterations that can occur innon-promoter sequences, and sometimes also can occur in sequences thatare not in the nucleotide sequence of interest, but in a relatednucleotide sequences (e.g., a gene involved in a different step of thesame pathway, a transport gene, and the like). Naturally occurringmutants sometimes can be found by isolating naturally occurring variantsfrom unique environments, in some embodiments.

In addition to the regulated promoter sequences, regulatory sequences,and coding polynucleotides provided herein, a nucleic acid reagent mayinclude a polynucleotide sequence 80% or more identical to the foregoing(or to the complementary sequences). That is, a nucleotide sequence thatis at least 80% or more, 81% or more, 82% or more, 83% or more, 84% ormore, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more,90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% ormore, 96% or more, 97% or more, 98% or more, or 99% or more identical toa nucleotide sequence described herein can be utilized. The term“identical” as used herein refers to two or more nucleotide sequenceshaving substantially the same nucleotide sequence when compared to eachother. One test for determining whether two nucleotide sequences oramino acids sequences are substantially identical is to determine thepercent of identical nucleotide sequences or amino acid sequencesshared.

Calculations of sequence identity can be performed as follows. Sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is sometimes 30% or more, 40% or more,50% or more, often 60% or more, and more often 70% or more, 80% or more,90% or more, or 100% of the length of the reference sequence. Thenucleotides or amino acids at corresponding nucleotide or polypeptidepositions, respectively, are then compared among the two sequences. Whena position in the first sequence is occupied by the same nucleotide oramino acid as the corresponding position in the second sequence, thenucleotides or amino acids are deemed to be identical at that position.The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, introduced foroptimal alignment of the two sequences.

Comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm.Percent identity between two amino acid or nucleotide sequences can bedetermined using the algorithm of Meyers & Miller, CABIOS 4: 11-17(1989), which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4. Also, percent identity between two amino acidsequences can be determined using the Needleman & Wunsch, J. Mol. Biol.48: 444-453 (1970) algorithm which has been incorporated into the GAPprogram in the GCG software package (available at the http addresswww.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and agap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. Percent identity between two nucleotide sequences can bedetermined using the GAP program in the GCG software package (availableat http address www.gcg.com), using a NWSgapdna.CMP matrix and a gapweight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or6. A set of parameters often used is a Blossum 62 scoring matrix with agap open penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

Sequence identity can also be determined by hybridization assaysconducted under stringent conditions. As use herein, the term “stringentconditions” refers to conditions for hybridization and washing.Stringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are describedin that reference and either can be used. An example of stringenthybridization conditions is hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions ishybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. Often,stringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C. More often, stringency conditionsare 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or morewashes at 0.2×SSC, 1% SDS at 65° C.

As noted above, nucleic acid reagents may also comprise one or more 5′UTR's, and one or more 3′UTR's. A 5′ UTR may comprise one or moreelements endogenous to the nucleotide sequence from which it originates,and sometimes includes one or more exogenous elements. A 5′ UTR canoriginate from any suitable nucleic acid, such as genomic DNA, plasmidDNA, RNA or mRNA, for example, from any suitable organism (e.g., virus,bacterium, yeast, fungi, plant, insect or mammal). The artisan mayselect appropriate elements for the 5′ UTR based upon the chosenexpression system (e.g., expression in a chosen organism, or expressionin a cell free system, for example). A 5′ UTR sometimes comprises one ormore of the following elements known to the artisan: enhancer sequences(e.g., transcriptional or translational), transcription initiation site,transcription factor binding site, translation regulation site,translation initiation site, translation factor binding site, accessoryprotein binding site, feedback regulation agent binding sites, Pribnowbox, TATA box, −35 element, E-box (helix-loop-helix binding element),ribosome binding site, replicon, internal ribosome entry site (IRES),silencer element and the like. In some embodiments, a promoter elementmay be isolated such that all 5′ UTR elements necessary for properconditional regulation are contained in the promoter element fragment,or within a functional subsequence of a promoter element fragment.

A 5′UTR in the nucleic acid reagent can comprise a translationalenhancer nucleotide sequence. A translational enhancer nucleotidesequence often is located between the promoter and the target nucleotidesequence in a nucleic acid reagent. A translational enhancer sequenceoften binds to a ribosome, sometimes is an 18S rRNA-bindingribonucleotide sequence (i.e., a 40S ribosome binding sequence) andsometimes is an internal ribosome entry sequence (IRES). An IRESgenerally forms an RNA scaffold with precisely placed RNA tertiarystructures that contact a 40S ribosomal subunit via a number of specificintermolecular interactions. Examples of ribosomal enhancer sequencesare known and can be identified by the artisan (e.g., Mignone et al.,Nucleic Acids Research 33: D141-D146 (2005); Paulous et al., NucleicAcids Research 31: 722-733 (2003); Akbergenov et al., Nucleic AcidsResearch 32: 239-247 (2004); Mignone et al., Genome Biology 3(3):reviews 0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:3401-3411 (2002); Shaloiko et al., http addresswww.interscience.wiley.com, DOI: 10.1002/bit.20267; and Gallie et al.,Nucleic Acids Research 15: 3257-3273 (1987)).

A translational enhancer sequence sometimes is a eukaryotic sequence,such as a Kozak consensus sequence or other sequence (e.g., hydroidpolyp sequence, GenBank accession no. U07128). A translational enhancersequence sometimes is a prokaryotic sequence, such as a Shine-Dalgarnoconsensus sequence. In certain embodiments, the translational enhancersequence is a viral nucleotide sequence. A translational enhancersequence sometimes is from a 5′ UTR of a plant virus, such as TobaccoMosaic Virus (TMV), Alfalfa Mosaic Virus (AMV); Tobacco Etch Virus(ETV); Potato Virus Y (PVY); Turnip Mosaic (poty) Virus and Pea SeedBorne Mosaic Virus, for example. In certain embodiments, an omegasequence about 67 bases in length from TMV is included in the nucleicacid reagent as a translational enhancer sequence (e.g., devoid ofguanosine nucleotides and includes a 25 nucleotide long poly (CAA)central region).

A 3′ UTR may comprise one or more elements endogenous to the nucleotidesequence from which it originates and sometimes includes one or moreexogenous elements. A 3′ UTR may originate from any suitable nucleicacid, such as genomic DNA, plasmid DNA, RNA or mRNA, for example, fromany suitable organism (e.g., a virus, bacterium, yeast, fungi, plant,insect or mammal). The artisan can select appropriate elements for the3′ UTR based upon the chosen expression system (e.g., expression in achosen organism, for example). A 3′ UTR sometimes comprises one or moreof the following elements known to the artisan: transcription regulationsite, transcription initiation site, transcription termination site,transcription factor binding site, translation regulation site,translation termination site, translation initiation site, translationfactor binding site, ribosome binding site, replicon, enhancer element,silencer element and polyadenosine tail. A 3′ UTR often includes apolyadenosine tail and sometimes does not, and if a polyadenosine tailis present, one or more adenosine moieties may be added or deleted fromit (e.g., about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45 or about 50 adenosine moieties may be addedor subtracted).

In some embodiments, modification of a 5′ UTR and/or a 3′ UTR can beused to alter (e.g., increase, add, decrease or substantially eliminate)the activity of a promoter. Alteration of the promoter activity can inturn alter the activity of a peptide, polypeptide or protein (e.g.,enzyme activity for example), by a change in transcription of thenucleotide sequence(s) of interest from an operably linked promoterelement comprising the modified 5′ or 3′ UTR. For example, amicroorganism can be engineered by genetic modification to express anucleic acid reagent comprising a modified 5′ or 3′ UTR that can add anovel activity (e.g., an activity not normally found in the hostorganism) or increase the expression of an existing activity byincreasing transcription from a homologous or heterologous promoteroperably linked to a nucleotide sequence of interest (e.g., homologousor heterologous nucleotide sequence of interest), in certainembodiments. In some embodiments, a microorganism can be engineered bygenetic modification to express a nucleic acid reagent comprising amodified 5′ or 3′ UTR that can decrease the expression of an activity bydecreasing or substantially eliminating transcription from a homologousor heterologous promoter operably linked to a nucleotide sequence ofinterest, in certain embodiments.

A nucleotide reagent sometimes can comprise a target nucleotidesequence. A “target nucleotide sequence” as used herein encodes anucleic acid, peptide, polypeptide or protein of interest, and may be aribonucleotide sequence or a deoxyribonucleotide sequence. A targetnucleic acid sometimes is an untranslated ribonucleic acid and sometimesis a translated ribonucleic acid. An untranslated ribonucleic acid mayinclude, but is not limited to, a small interfering ribonucleic acid(siRNA), a short hairpin ribonucleic acid (shRNA), other ribonucleicacid capable of RNA interference (RNAi), an antisense ribonucleic acid,or a ribozyme. A translatable target nucleotide sequence (e.g., a targetribonucleotide sequence) sometimes encodes a peptide, polypeptide orprotein, which are sometimes referred to herein as “target peptides,”“target polypeptides” or “target proteins.”

Any peptides, polypeptides or proteins, or an activity catalyzed by oneor more peptides, polypeptides or proteins may be encoded by a targetnucleotide sequence and may be selected by a user. Representativeproteins include enzymes (e.g., hexanoate synthase, thioesterase,monooxygenase, monooxygenase reductase, fatty alcohol oxidase,6-oxohexanoic acid dehydrogenase, 6-hydroxyhexanoic acid dehydrogenaseand the like, for example), antibodies, serum proteins (e.g., albumin),membrane bound proteins, hormones (e.g., growth hormone, erythropoietin,insulin, etc.), cytokines, etc., and include both naturally occurringand exogenously expressed polypeptides. Representative activities (e.g.,enzymes or combinations of enzymes which are functionally associated toprovide an activity) include hexanoate synthase activity, thioesteraseactivity, monooxygenase activity, 6-oxohexanoic acid dehydrogenaseactivity, 6-hydroxyhexanoic acid dehydrogenase activity, beta-oxidationactivity and the like, for example. The term “enzyme” as used hereinrefers to a protein which can act as a catalyst to induce a chemicalchange in other compounds, thereby producing one or more products fromone or more substrates.

Specific polypeptides (e.g., enzymes) useful for embodiments describedherein are listed herein. The term “protein” as used herein refers to amolecule having a sequence of amino acids linked by peptide bonds. Thisterm includes fusion proteins, oligopeptides, peptides, cyclic peptides,polypeptides and polypeptide derivatives, whether native or recombinant,and also includes fragments, derivatives, homologs, and variantsthereof. A protein or polypeptide sometimes is of intracellular origin(e.g., located in the nucleus, cytosol, or interstitial space of hostcells in vivo) and sometimes is a cell membrane protein in vivo. In someembodiments (described above, and in further detail hereafter inEngineering and Alteration Methods), a genetic modification can resultin a modification (e.g., increase, substantially increase, decrease orsubstantially decrease) of a target activity.

A translatable nucleotide sequence generally is located between a startcodon (AUG in ribonucleic acids and ATG in deoxyribonucleic acids) and astop codon (e.g., UAA (ochre), UAG (amber) or UGA (opal) in ribonucleicacids and TAA, TAG or TGA in deoxyribonucleic acids), and sometimes isreferred to herein as an “open reading frame” (ORF). A translatablenucleotide sequence (e.g., ORF) sometimes is encoded differently in oneorganism (e.g., most organisms encode CTG as leucine) than in anotherorganism (e.g., C. tropicalis encodes CTG as serine). In someembodiments, a translatable nucleotide sequence is altered to correctalternate genetic code (e.g., codon usage) differences between anucleotide donor organism and an nucleotide recipient organism (e.g.,engineered organism). In certain embodiments, a translatable nucleotidesequence is altered to improve; (i) codon usage, (ii) transcriptionalefficiency, (iii) translational efficiency, (iv) the like, andcombinations thereof.

A nucleic acid reagent sometimes comprises one or more ORFs. An ORF maybe from any suitable source, sometimes from genomic DNA, mRNA, reversetranscribed RNA or complementary DNA (cDNA) or a nucleic acid librarycomprising one or more of the foregoing, and is from any organismspecies that contains a nucleic acid sequence of interest, protein ofinterest, or activity of interest. Non-limiting examples of organismsfrom which an ORF can be obtained include bacteria, yeast, fungi, human,insect, nematode, bovine, equine, canine, feline, rat or mouse, forexample.

A nucleic acid reagent sometimes comprises a nucleotide sequenceadjacent to an ORF that is translated in conjunction with the ORF andencodes an amino acid tag. The tag-encoding nucleotide sequence islocated 3′ and/or 5′ of an ORF in the nucleic acid reagent, therebyencoding a tag at the C-terminus or N-terminus of the protein or peptideencoded by the ORF. Any tag that does not abrogate in vitrotranscription and/or translation may be utilized and may beappropriately selected by the artisan. Tags may facilitate isolationand/or purification of the desired ORF product from culture orfermentation media.

A tag sometimes specifically binds a molecule or moiety of a solid phaseor a detectable label, for example, thereby having utility forisolating, purifying and/or detecting a protein or peptide encoded bythe ORF. In some embodiments, a tag comprises one or more of thefollowing elements: FLAG (e.g., DYKDDDDKG), V5 (e.g., GKPIPNPLLGLDST),c-MYC (e.g., EQKLISEEDL), HSV (e.g., QPELAPEDPED), influenzahemaglutinin, HA (e.g., YPYDVPDYA), VSV-G (e.g., YTDIEMNRLGK), bacterialglutathione-5-transferase, maltose binding protein, a streptavidin- oravidin-binding tag (e.g., pcDNA™6 BioEase™ Gateway® Biotinylation System(Invitrogen)), thioredoxin, β-galactosidase, VSV-glycoprotein, afluorescent protein (e.g., green fluorescent protein or one of its manycolor variants (e.g., yellow, red, blue)), a polylysine or polyargininesequence, a polyhistidine sequence (e.g., His6) or other sequence thatchelates a metal (e.g., cobalt, zinc, copper), and/or a cysteine-richsequence that binds to an arsenic-containing molecule. In certainembodiments, a cysteine-rich tag comprises the amino acid sequenceCC-Xn-CC, wherein X is any amino acid and n is 1 to 3, and thecysteine-rich sequence sometimes is CCPGCC. In certain embodiments, thetag comprises a cysteine-rich element and a polyhistidine element (e.g.,CCPGCC and His6).

A tag often conveniently binds to a binding partner. For example, sometags bind to an antibody (e.g., FLAG) and sometimes specifically bind toa small molecule. For example, a polyhistidine tag specifically chelatesa bivalent metal, such as copper, zinc and cobalt; a polylysine orpolyarginine tag specifically binds to a zinc finger; a glutathioneS-transferase tag binds to glutathione; and a cysteine-rich tagspecifically binds to an arsenic-containing molecule. Arsenic-containingmolecules include LUMIO™ agents (Invitrogen, California), such as FlAsH™(EDT2[4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2])and ReAsH reagents (e.g., U.S. Pat. No. 5,932,474 to Tsien et al.,entitled “Target Sequences for Synthetic Molecules;” U.S. Pat. No.6,054,271 to Tsien et al., entitled “Methods of Using SyntheticMolecules and Target Sequences;” U.S. Pat. Nos. 6,451,569 and 6,008,378;published U.S. Patent Application 2003/0083373, and published PCT PatentApplication WO 99/21013, all to Tsien et al. and all entitled “SyntheticMolecules that Specifically React with Target Sequences”). Suchantibodies and small molecules sometimes are linked to a solid phase forconvenient isolation of the target protein or target peptide.

A tag sometimes comprises a sequence that localizes a translated proteinor peptide to a component in a system, which is referred to as a “signalsequence” or “localization signal sequence” herein. A signal sequenceoften is incorporated at the N-terminus of a target protein or targetpeptide, and sometimes is incorporated at the C-terminus. Examples ofsignal sequences are known to the artisan, are readily incorporated intoa nucleic acid reagent, and often are selected according to the organismin which expression of the nucleic acid reagent is performed. A signalsequence in some embodiments localizes a translated protein or peptideto a cell membrane. Examples of signal sequences include, but are notlimited to, a nucleus targeting signal (e.g., steroid receptor sequenceand N-terminal sequence of SV40 virus large T antigen); mitochondrialtargeting signal (e.g., amino acid sequence that forms an amphipathichelix); peroxisome targeting signal (e.g., C-terminal sequence in YFGfrom S. cerevisiae); and a secretion signal (e.g., N-terminal sequencesfrom invertase, mating factor alpha, PHO5 and SUC2 in S. cerevisiae;multiple N-terminal sequences of B. subtilis proteins (e.g., Tjalsma etal., Microbiol. Molec. Biol. Rev. 64: 515-547 (2000)); alpha amylasesignal sequence (e.g., U.S. Pat. No. 6,288,302); pectate lyase signalsequence (e.g., U.S. Pat. No. 5,846,818); precollagen signal sequence(e.g., U.S. Pat. No. 5,712,114); OmpA signal sequence (e.g., U.S. Pat.No. 5,470,719); lam beta signal sequence (e.g., U.S. Pat. No.5,389,529); B. brevis signal sequence (e.g., U.S. Pat. No. 5,232,841);and P. pastoris signal sequence (e.g., U.S. Pat. No. 5,268,273)).

A tag sometimes is directly adjacent to the amino acid sequence encodedby an ORF (i.e., there is no intervening sequence) and sometimes a tagis substantially adjacent to an ORF encoded amino acid sequence (e.g.,an intervening sequence is present). An intervening sequence sometimesincludes a recognition site for a protease, which is useful for cleavinga tag from a target protein or peptide. In some embodiments, theintervening sequence is cleaved by Factor Xa (e.g., recognition site I(E/D)GR), thrombin (e.g., recognition site LVPRGS), enterokinase (e.g.,recognition site DDDDK), TEV protease (e.g., recognition site ENLYFQG)or PreScission™ protease (e.g., recognition site LEVLFQGP), for example.

An intervening sequence sometimes is referred to herein as a “linkersequence,” and may be of any suitable length selected by the artisan. Alinker sequence sometimes is about 1 to about 20 amino acids in length,and sometimes about 5 to about 10 amino acids in length. The artisan mayselect the linker length to substantially preserve target protein orpeptide function (e.g., a tag may reduce target protein or peptidefunction unless separated by a linker), to enhance disassociation of atag from a target protein or peptide when a protease cleavage site ispresent (e.g., cleavage may be enhanced when a linker is present), andto enhance interaction of a tag/target protein product with a solidphase. A linker can be of any suitable amino acid content, and oftencomprises a higher proportion of amino acids having relatively shortside chains (e.g., glycine, alanine, serine and threonine).

A nucleic acid reagent sometimes includes a stop codon between a tagelement and an insertion element or ORF, which can be useful fortranslating an ORF with or without the tag. Mutant tRNA molecules thatrecognize stop codons (described above) suppress translation terminationand thereby are designated “suppressor tRNAs.” Suppressor tRNAs canresult in the insertion of amino acids and continuation of translationpast stop codons (e.g., U.S. Patent Application No. 60/587,583, filedJul. 14, 2004, entitled “Production of Fusion Proteins by Cell-FreeProtein Synthesis,”; Eggertsson, et al., (1988) Microbiological Review52(3):354-374, and Engleerg-Kukla, et al. (1996) in Escherichia coli andSalmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,Neidhardt, et al. eds., ASM Press, Washington, D.C.). A number ofsuppressor tRNAs are known, including but not limited to, supE, supP,supD, supF and supZ suppressors, which suppress the termination oftranslation of the amber stop codon; supB, glT, supL, supN, supC andsupM suppressors, which suppress the function of the ochre stop codonand glyT, trpT and Su-9 suppressors, which suppress the function of theopal stop codon. In general, suppressor tRNAs contain one or moremutations in the anti-codon loop of the tRNA that allows the tRNA tobase pair with a codon that ordinarily functions as a stop codon. Themutant tRNA is charged with its cognate amino acid residue and thecognate amino acid residue is inserted into the translating polypeptidewhen the stop codon is encountered. Mutations that enhance theefficiency of termination suppressors (i.e., increase stop codonread-through) have been identified. These include, but are not limitedto, mutations in the uar gene (also known as the prfA gene), mutationsin the ups gene, mutations in the sueA, sueB and sueC genes, mutationsin the rpsD (ramA) and rpsE (spcA) genes and mutations in the rplL gene.

Thus, a nucleic acid reagent comprising a stop codon located between anORF and a tag can yield a translated ORF alone when no suppressor tRNAis present in the translation system, and can yield a translated ORF-tagfusion when a suppressor tRNA is present in the system. Suppressor tRNAcan be generated in cells transfected with a nucleic acid encoding thetRNA (e.g., a replication incompetent adenovirus containing the humantRNA-Ser suppressor gene can be transfected into cells, or a YACcontaining a yeast or bacterial tRNA suppressor gene can be transfectedinto yeast cells, for example). Vectors for synthesizing suppressor tRNAand for translating ORFs with or without a tag are available to theartisan (e.g., Tag-On-Demand™ kit (Invitrogen Corporation, California);Tag-On-Demand™ Suppressor Supernatant Instruction Manual, Version B, 6Jun. 2003, at http addresswww.invitrogen.com/content/sfs/manuals/tagondemand_supernatant_man.pdf;Tag-On-Demand™ Gateway® Vector Instruction Manual, Version B, 20 Jun.,2003 at http addresswww.invitrogen.com/content/sfs/manuals/tagondemand_vectors_man.pdf; andCapone et al., Amber, ochre and opal suppressor tRNA genes derived froma human serine tRNA gene. EMBO J. 4:213, 1985).

Any convenient cloning strategy known in the art may be utilized toincorporate an element, such as an ORF, into a nucleic acid reagent.Known methods can be utilized to insert an element into the templateindependent of an insertion element, such as (1) cleaving the templateat one or more existing restriction enzyme sites and ligating an elementof interest and (2) adding restriction enzyme sites to the template byhybridizing oligonucleotide primers that include one or more suitablerestriction enzyme sites and amplifying by polymerase chain reaction(described in greater detail herein). Other cloning strategies takeadvantage of one or more insertion sites present or inserted into thenucleic acid reagent, such as an oligonucleotide primer hybridizationsite for PCR, for example, and others described herein. In someembodiments, a cloning strategy can be combined with geneticmanipulation such as recombination (e.g., recombination of a nucleicacid reagent with a nucleic acid sequence of interest into the genome ofthe organism to be modified, as described further herein). In someembodiments, the cloned ORF(s) can produce (directly or indirectly)adipic acid, by engineering a microorganism with one or more ORFs ofinterest, which microorganism comprises one or more altered activitiesselected from the group consisting of 6-oxohexanoic acid dehydrogenaseactivity, 6-hydroxyhexanoic acid dehydrogenase activity, hexanoatesynthase activity and monooxygenase activity.

In some embodiments, the nucleic acid reagent includes one or morerecombinase insertion sites. A recombinase insertion site is arecognition sequence on a nucleic acid molecule that participates in anintegration/recombination reaction by recombination proteins. Forexample, the recombination site for Cre recombinase is IoxP, which is a34 base pair sequence comprised of two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence (e.g., FIG. 1 of Sauer, B., Curr. Opin. Biotech. 5:521-527(1994)). Other examples of recombination sites include attB, attP, attL,and attR sequences, and mutants, fragments, variants and derivativesthereof, which are recognized by the recombination protein λ Int and bythe auxiliary proteins integration host factor (IHF), FIS andexcisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732; 6,143,557; 6,171,861;6,270,969; 6,277,608; and 6,720,140; U.S. patent application Ser. Nos.09/517,466, filed Mar. 2, 2000, and 09/732,914, filed Aug. 14, 2003, andin U.S. patent publication no. 2002-0007051-A1; Landy, Curr. Opin.Biotech. 3:699-707 (1993)).

Examples of recombinase cloning nucleic acids are in Gateway® systems(Invitrogen, California), which include at least one recombination sitefor cloning a desired nucleic acid molecules in vivo or in vitro. Insome embodiments, the system utilizes vectors that contain at least twodifferent site-specific recombination sites, often based on thebacteriophage lambda system (e.g., att1 and att2), and are mutated fromthe wild-type (att0) sites. Each mutated site has a unique specificityfor its cognate partner att site (i.e., its binding partnerrecombination site) of the same type (for example attB1 with attP1, orattL1 with attR1) and will not cross-react with recombination sites ofthe other mutant type or with the wild-type att0 site. Different sitespecificities allow directional cloning or linkage of desired moleculesthus providing desired orientation of the cloned molecules. Nucleic acidfragments flanked by recombination sites are cloned and subcloned usingthe Gateway® system by replacing a selectable marker (for example, ccdB)flanked by att sites on the recipient plasmid molecule, sometimes termedthe Destination Vector. Desired clones are then selected bytransformation of a ccdB sensitive host strain and positive selectionfor a marker on the recipient molecule. Similar strategies for negativeselection (e.g., use of toxic genes) can be used in other organisms suchas thymidine kinase (TK) in mammals and insects.

A recombination system useful for engineering yeast is outlined briefly.The system makes use of the URA3 gene (e.g., for S. cerevisiae and C.albicans, for example) or URA4 and URA5 genes (e.g., for S. pombe, forexample) and toxicity of the nucleotide analogue 5-Fluoroorotic acid(5-FOA). The URA3 or URA4 and URA5 genes encode orotine-5′-monophosphate(OMP) decarboxylase. Yeast with an active URA3 or URA4 and URA5 gene(phenotypically Ura+) convert 5-FOA to fluorodeoxyuridine, which istoxic to yeast cells. Yeast carrying a mutation in the appropriategene(s) or having a knock out of the appropriate gene(s) can grow in thepresence of 5-FOA, if the media is also supplemented with uracil.

A nucleic acid engineering construct can be made which may comprise theURA3 gene or cassette (for S. cerevisiae), flanked on either side by thesame nucleotide sequence in the same orientation. The URA3 cassettecomprises a promoter, the URA3 gene and a functional transcriptionterminator. Target sequences which direct the construct to a particularnucleic acid region of interest in the organism to be engineered areadded such that the target sequences are adjacent to and abut theflanking sequences on either side of the URA3 cassette. Yeast can betransformed with the engineering construct and plated on minimal mediawithout uracil. Colonies can be screened by PCR to determine thosetransformants that have the engineering construct inserted in the properlocation in the genome. Checking insertion location prior to selectingfor recombination of the ura3 cassette may reduce the number ofincorrect clones carried through to later stages of the procedure.Correctly inserted transformants can then be replica plated on minimalmedia containing 5-FOA to select for recombination of the URA3 cassetteout of the construct, leaving a disrupted gene and an identifiablefootprint (e.g., nucleic acid sequence) that can be use to verify thepresence of the disrupted gene. The technique described is useful fordisrupting or “knocking out” gene function, but also can be used toinsert genes or constructs into a host organisms genome in a targeted,sequence specific manner.

In certain embodiments, a nucleic acid reagent includes one or moretopoisomerase insertion sites. A topoisomerase insertion site is adefined nucleotide sequence recognized and bound by a site-specifictopoisomerase. For example, the nucleotide sequence 5′-(C/T)CCTT-3′ is atopoisomerase recognition site bound specifically by most poxvirustopoisomerases, including vaccinia virus DNA topoisomerase I. Afterbinding to the recognition sequence, the topoisomerase cleaves thestrand at the 3′-most thymidine of the recognition site to produce anucleotide sequence comprising 5′-(C/T)CCTT-PO4-TOPO, a complex of thetopoisomerase covalently bound to the 3′ phosphate via a tyrosine in thetopoisomerase (e.g., Shuman, J. Biol. Chem. 266:11372-11379, 1991;Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; U.S. Pat. No.5,766,891; PCT/US95/16099; and PCT/US98/12372). In comparison, thenucleotide sequence 5′-GCAACTT-3′ is a topoisomerase recognition sitefor type IA E. coli topoisomerase III. An element to be inserted oftenis combined with topoisomerase-reacted template and thereby incorporatedinto the nucleic acid reagent (e.g., World Wide Web URLinvitrogen.com/downloads/F-13512_Topo_Flyer.pdf; World Wide Web URLinvitrogen.com/content/sfs/brochures/710_(—)021849%20_B_TOPOCloning_bro.pdf;TOPO TA Cloning® Kit and Zero Blunt® TOPO® Cloning Kit productinformation).

A nucleic acid reagent sometimes contains one or more origin ofreplication (ORI) elements. In some embodiments, a template comprisestwo or more ORIs, where one functions efficiently in one organism (e.g.,a bacterium) and another functions efficiently in another organism(e.g., a eukaryote, like yeast for example). In some embodiments, an ORImay function efficiently in one species (e.g., S. cerevisiae, forexample) and another ORI may function efficiently in a different species(e.g., S. pombe, for example). A nucleic acid reagent also sometimesincludes one or more transcription regulation sites.

A nucleic acid reagent can include one or more selection elements (e.g.,elements for selection of the presence of the nucleic acid reagent, andnot for activation of a promoter element which can be selectivelyregulated). Selection elements often are utilized using known processesto determine whether a nucleic acid reagent is included in a cell. Insome embodiments, a nucleic acid reagent includes two or more selectionelements, where one functions efficiently in one organism and anotherfunctions efficiently in another organism. Examples of selectionelements include, but are not limited to, (1) nucleic acid segments thatencode products that provide resistance against otherwise toxiccompounds (e.g., antibiotics); (2) nucleic acid segments that encodeproducts that are otherwise lacking in the recipient cell (e.g.,essential products, tRNA genes, auxotrophic markers); (3) nucleic acidsegments that encode products that suppress the activity of a geneproduct; (4) nucleic acid segments that encode products that can bereadily identified (e.g., phenotypic markers such as antibiotics (e.g.,β-lactamase), β-galactosidase, green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), cyanfluorescent protein (CFP), and cell surface proteins); (5) nucleic acidsegments that bind products that are otherwise detrimental to cellsurvival and/or function; (6) nucleic acid segments that otherwiseinhibit the activity of any of the nucleic acid segments described inNos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acidsegments that bind products that modify a substrate (e.g., restrictionendonucleases); (8) nucleic acid segments that can be used to isolate oridentify a desired molecule (e.g., specific protein binding sites); (9)nucleic acid segments that encode a specific nucleotide sequence thatcan be otherwise non-functional (e.g., for PCR amplification ofsubpopulations of molecules); (10) nucleic acid segments that, whenabsent, directly or indirectly confer resistance or sensitivity toparticular compounds; (11) nucleic acid segments that encode productsthat either are toxic or convert a relatively non-toxic compound to atoxic compound (e.g., Herpes simplex thymidine kinase, cytosinedeaminase) in recipient cells; (12) nucleic acid segments that inhibitreplication, partition or heritability of nucleic acid molecules thatcontain them; and/or (13) nucleic acid segments that encode conditionalreplication functions, e.g., replication in certain hosts or host cellstrains or under certain environmental conditions (e.g., temperature,nutritional conditions, and the like).

A nucleic acid reagent is of any form useful for in vivo transcriptionand/or translation. A nucleic acid sometimes is a plasmid, such as asupercoiled plasmid, sometimes is a yeast artificial chromosome (e.g.,YAC), sometimes is a linear nucleic acid (e.g., a linear nucleic acidproduced by PCR or by restriction digest), sometimes is single-strandedand sometimes is double-stranded. A nucleic acid reagent sometimes isprepared by an amplification process, such as a polymerase chainreaction (PCR) process or transcription-mediated amplification process(TMA). In TMA, two enzymes are used in an isothermal reaction to produceamplification products detected by light emission (see, e.g.,Biochemistry 1996 Jun. 25; 35(25):8429-38 and http addresswww.devicelink.com/ivdt/archive/00/11/007.html). Standard PCR processesare known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and5,656,493), and generally are performed in cycles. Each cycle includesheat denaturation, in which hybrid nucleic acids dissociate; cooling, inwhich primer oligonucleotides hybridize; and extension of theoligonucleotides by a polymerase (i.e., Taq polymerase). An example of aPCR cyclical process is treating the sample at 95° C. for 5 minutes;repeating forty-five cycles of 95° C. for 1 minute, 59° C. for 1 minute,10 seconds, and 72° C. for 1 minute 30 seconds; and then treating thesample at 72° C. for 5 minutes. Multiple cycles frequently are performedusing a commercially available thermal cycler. PCR amplificationproducts sometimes are stored for a time at a lower temperature (e.g.,at 4° C.) and sometimes are frozen (e.g., at −20° C.) before analysis.

In some embodiments, a nucleic acid reagent, protein reagent, proteinfragment reagent or other reagent described herein is isolated orpurified. The term “isolated” as used herein refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring, or a host cell if expressed exogenously), and thusis altered “by the hand of man” from its original environment. The term“purified” as used herein with reference to molecules does not refer toabsolute purity. Rather, “purified” refers to a substance in acomposition that contains fewer substance species in the same class(e.g., nucleic acid or protein species) other than the substance ofinterest in comparison to the sample from which it originated.“Purified,” if a nucleic acid or protein for example, refers to asubstance in a composition that contains fewer nucleic acid species orprotein species other than the nucleic acid or protein of interest incomparison to the sample from which it originated. Sometimes, a proteinor nucleic acid is “substantially pure,” indicating that the protein ornucleic acid represents at least 50% of protein or nucleic acid on amass basis of the composition. Often, a substantially pure protein ornucleic acid is at least 75% on a mass basis of the composition, andsometimes at least 95% on a mass basis of the composition.

Engineering and Alteration Methods

Methods and compositions (e.g., nucleic acid reagents) described hereincan be used to generate engineered microorganisms. As noted above, theterm “engineered microorganism” as used herein refers to a modifiedorganism that includes one or more activities distinct from an activitypresent in a microorganism utilized as a starting point for modification(e.g., host microorganism or unmodified organism). Engineeredmicroorganisms typically arise as a result of a genetic modification,usually introduced or selected for, by one of skill in the art usingreadily available techniques. Non-limiting examples of methods usefulfor generating an altered activity include, introducing a heterologouspolynucleotide (e.g., nucleic acid or gene integration, also referred toas “knock in”), removing an endogenous polynucleotide, altering thesequence of an existing endogenous nucleic acid sequence (e.g.,site-directed mutagenesis), disruption of an existing endogenous nucleicacid sequence (e.g., knock outs and transposon or insertion elementmediated mutagenesis), selection for an altered activity where theselection causes a change in a naturally occurring activity that can bestably inherited (e.g., causes a change in a nucleic acid sequence inthe genome of the organism or in an epigenetic nucleic acid that isreplicated and passed on to daughter cells), PCR-based mutagenesis, andthe like. The term “mutagenesis” as used herein refers to anymodification to a nucleic acid (e.g., nucleic acid reagent, or hostchromosome, for example) that is subsequently used to generate a productin a host or modified organism. Non-limiting examples of mutagenesisinclude, deletion, insertion, substitution, rearrangement, pointmutations, suppressor mutations and the like. Mutagenesis methods areknown in the art and are readily available to the artisan. Non-limitingexamples of mutagenesis methods are described herein and can also befound in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) MolecularCloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Another non-limiting example of mutagenesis can beconducted using a Stratagene (San Diego, Calif.) “QuickChange” kitaccording to the manufacturer's instructions.

The term “genetic modification” as used herein refers to any suitablenucleic acid addition, removal or alteration that facilitates productionof a target product (e.g., adipic acid, 6-hydroxyhexanoic acid) in anengineered microorganism. Genetic modifications include, withoutlimitation, insertion of one or more nucleotides in a native nucleicacid of a host organism in one or more locations, deletion of one ormore nucleotides in a native nucleic acid of a host organism in one ormore locations, modification or substitution of one or more nucleotidesin a native nucleic acid of a host organism in one or more locations,insertion of a non-native nucleic acid into a host organism (e.g.,insertion of an autonomously replicating vector), and removal of anon-native nucleic acid in a host organism (e.g., removal of a vector).

The term “heterologous polynucleotide” as used herein refers to anucleotide sequence not present in a host microorganism in someembodiments. In certain embodiments, a heterologous polynucleotide ispresent in a different amount (e.g., different copy number) than in ahost microorganism, which can be accomplished, for example, byintroducing more copies of a particular nucleotide sequence to a hostmicroorganism (e.g., the particular nucleotide sequence may be in anucleic acid autonomous of the host chromosome or may be inserted into achromosome). A heterologous polynucleotide is from a different organismin some embodiments, and in certain embodiments, is from the same typeof organism but from an outside source (e.g., a recombinant source).

In some embodiments, an organism engineered using the methods andnucleic acid reagents described herein can produce adipic acid. Incertain embodiments, an engineered microorganism described herein thatproduces adipic acid may comprise one or more altered activitiesselected from the group consisting of 6-oxohexanoic acid dehydrogenaseactivity, omega oxo fatty acid dehydrogenase activity, 6-hydroxyhexanoicacid dehydrogenase activity, omega hydroxyl fatty acid dehydrogenaseactivity, hexanoate synthase activity and monooxygenase activity. Insome embodiments, an engineered microorganism as described herein maycomprise a genetic modification that adds or increases the 6-oxohexanoicacid dehydrogenase activity, omega oxo fatty acid dehydrogenaseactivity, 6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxylfatty acid dehydrogenase activity, hexanoate synthase activity andmonooxygenase activity.

In certain embodiments, an engineered microorganism described herein cancomprise an altered thioesterase activity. In some embodiments, theengineered microorganism may comprise a genetic alteration that adds orincreases a thioesterase activity. In some embodiments, the engineeredmicroorganism comprising a genetic alteration that adds or increases athioesterase activity, may further comprise a heterologouspolynucleotide encoding a polypeptide having thioesterase activity.

The term “altered activity” as used herein refers to an activity in anengineered microorganism that is added or modified relative to the hostmicroorganism (e.g., added, increased, reduced, inhibited or removedactivity). An activity can be altered by introducing a geneticmodification to a host microorganism that yields an engineeredmicroorganism having added, increased, reduced, inhibited or removedactivity.

An added activity often is an activity not detectable in a hostmicroorganism. An increased activity generally is an activity detectablein a host microorganism that has been increased in an engineeredmicroorganism. An activity can be increased to any suitable level forproduction of a target product (e.g., adipic acid, 6-hydroxyhexanoicacid), including but not limited to less than 2-fold (e.g., about 10%increase to about 99% increase; about 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% increase), 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, of 10-fold increase, or greater than about 10-fold increase. Areduced or inhibited activity generally is an activity detectable in ahost microorganism that has been reduced or inhibited in an engineeredmicroorganism. An activity can be reduced to undetectable levels in someembodiments, or detectable levels in certain embodiments. An activitycan be decreased to any suitable level for production of a targetproduct (e.g., adipic acid, 6-hydroxyhexanoic acid), including but notlimited to less than 2-fold (e.g., about 10% decrease to about 99%decrease; about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% decrease),2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of10-fold decrease, or greater than about 10-fold decrease.

An altered activity sometimes is an activity not detectable in a hostorganism and is added to an engineered organism. An altered activityalso may be an activity detectable in a host organism and is increasedin an engineered organism. An activity may be added or increased byincreasing the number of copies of a polynucleotide that encodes apolypeptide having a target activity, in some embodiments. In certainembodiments an activity can be added or increased by inserting into ahost microorganism a heterologous polynucleotide that encodes apolypeptide having the added activity. In certain embodiments, anactivity can be added or increased by inserting into a hostmicroorganism a heterologous polynucleotide that is (i) operably linkedto another polynucleotide that encodes a polypeptide having the addedactivity, and (ii) up regulates production of the polynucleotide. Thus,an activity can be added or increased by inserting or modifying aregulatory polynucleotide operably linked to another polynucleotide thatencodes a polypeptide having the target activity. In certainembodiments, an activity can be added or increased by subjecting a hostmicroorganism to a selective environment and screening formicroorganisms that have a detectable level of the target activity.Examples of a selective environment include, without limitation, amedium containing a substrate that a host organism can process and amedium lacking a substrate that a host organism can process.

An altered activity sometimes is an activity detectable in a hostorganism and is reduced, inhibited or removed (i.e., not detectable) inan engineered organism. An activity may be reduced or removed bydecreasing the number of copies of a polynucleotide that encodes apolypeptide having a target activity, in some embodiments. In someembodiments, an activity can be reduced or removed by (i) inserting apolynucleotide within a polynucleotide that encodes a polypeptide havingthe target activity (disruptive insertion), and/or (ii) removing aportion of or all of a polynucleotide that encodes a polypeptide havingthe target activity (deletion or knock out, respectively). In certainembodiments, an activity can be reduced or removed by inserting into ahost microorganism a heterologous polynucleotide that is (i) operablylinked to another polynucleotide that encodes a polypeptide having thetarget activity, and (ii) down regulates production of thepolynucleotide. Thus, an activity can be reduced or removed by insertingor modifying a regulatory polynucleotide operably linked to anotherpolynucleotide that encodes a polypeptide having the target activity.

An activity also can be reduced or removed by (i) inhibiting apolynucleotide that encodes a polypeptide having the activity or (ii)inhibiting a polynucleotide operably linked to another polynucleotidethat encodes a polypeptide having the activity. A polynucleotide can beinhibited by a suitable technique known in the art, such as bycontacting an RNA encoded by the polynucleotide with a specificinhibitory RNA (e.g., RNAi, siRNA, ribozyme). An activity also can bereduced or removed by contacting a polypeptide having the activity witha molecule that specifically inhibits the activity (e.g., enzymeinhibitor, antibody). In certain embodiments, an activity can be reducedor removed by subjecting a host microorganism to a selective environmentand screening for microorganisms that have a reduced level or removal ofthe target activity.

In some embodiments, an untranslated ribonucleic acid, or a cDNA can beused to reduce the expression of a particular activity or enzyme. Forexample, a microorganism can be engineered by genetic modification toexpress a nucleic acid reagent that reduces the expression of anactivity by producing an RNA molecule that is partially or substantiallyhomologous to a nucleic acid sequence of interest which encodes theactivity of interest. The RNA molecule can bind to the nucleic acidsequence of interest and inhibit the nucleic acid sequence fromperforming its natural function, in certain embodiments. In someembodiments, the RNA may alter the nucleic acid sequence of interestwhich encodes the activity of interest in a manner that the nucleic acidsequence of interest is no longer capable of performing its naturalfunction (e.g., the action of a ribozyme for example).

In certain embodiments, nucleotide sequences sometimes are added to,modified or removed from one or more of the nucleic acid reagentelements, such as the promoter, 5′UTR, target sequence, or 3′UTRelements, to enhance, potentially enhance, reduce, or potentially reducetranscription and/or translation before or after such elements areincorporated in a nucleic acid reagent. In some embodiments, one or moreof the following sequences may be modified or removed if they arepresent in a 5′UTR: a sequence that forms a stable secondary structure(e.g., quadruplex structure or stem loop stem structure (e.g., EMBLsequences X12949, AF274954, AF139980, AF152961, S95936, U194144,AF116649 or substantially identical sequences that form such stem loopstem structures)); a translation initiation codon upstream of the targetnucleotide sequence start codon; a stop codon upstream of the targetnucleotide sequence translation initiation codon; an ORF upstream of thetarget nucleotide sequence translation initiation codon; an ironresponsive element (IRE) or like sequence; and a 5′ terminaloligopyrimidine tract (TOP, e.g., consisting of 5-15 pyrimidinesadjacent to the cap). A translational enhancer sequence and/or aninternal ribosome entry site (IRES) sometimes is inserted into a 5′UTR(e.g., EMBL nucleotide sequences J04513, X87949, M95825, M12783,AF025841, AF013263, AF006822, M17169, M13440, M22427, D14838 and M17446and substantially identical nucleotide sequences). An AU-rich element(ARE, e.g., AUUUA repeats) and/or splicing junction that follows anon-sense codon sometimes is removed from or modified in a 3′UTR. Apolyadenosine tail sometimes is inserted into a 3′UTR if none ispresent, sometimes is removed if it is present, and adenosine moietiessometimes are added to or removed from a polyadenosine tail present in a3′UTR. Thus, some embodiments are directed to a process comprising:determining whether any nucleotide sequences that increase, potentiallyincrease, reduce or potentially reduce translation efficiency arepresent in the elements, and adding, removing or modifying one or moreof such sequences if they are identified. Certain embodiments aredirected to a process comprising: determining whether any nucleotidesequences that increase or potentially increase translation efficiencyare not present in the elements, and incorporating such sequences intothe nucleic acid reagent.

In some embodiments, an activity can be altered by modifying thenucleotide sequence of an ORF. An ORF sometimes is mutated or modified(for example, by point mutation, deletion mutation, insertion mutation,PCR based mutagenesis and the like) to alter, enhance or increase,reduce, substantially reduce or eliminate the activity of the encodedprotein or peptide. The protein or peptide encoded by a modified ORFsometimes is produced in a lower amount or may not be produced atdetectable levels, and in other embodiments, the product or proteinencoded by the modified ORF is produced at a higher level (e.g., codonssometimes are modified so they are compatible with tRNA's preferentiallyused in the host organism or engineered organism). To determine therelative activity, the activity from the product of the mutated ORF (orcell containing it) can be compared to the activity of the product orprotein encoded by the unmodified ORF (or cell containing it).

In some embodiments, an ORF nucleotide sequence sometimes is mutated ormodified to alter the triplet nucleotide sequences used to encode aminoacids (e.g., amino acid codon triplets, for example). Modification ofthe nucleotide sequence of an ORF to alter codon triplets sometimes isused to change the codon found in the original sequence to better matchthe preferred codon usage of the organism in which the ORF or nucleicacid reagent will be expressed. The codon usage, and therefore the codontriplets encoded by a nucleic acid sequence, in bacteria may bedifferent from the preferred codon usage in eukaryotes, like yeast orplants for example. Preferred codon usage also may be different betweenbacterial species. In certain embodiments an ORF nucleotide sequencessometimes is modified to eliminate codon pairs and/or eliminate mRNAsecondary structures that can cause pauses during translation of themRNA encoded by the ORF nucleotide sequence. Translational pausingsometimes occurs when nucleic acid secondary structures exist in anmRNA, and sometimes occurs due to the presence of codon pairs that slowthe rate of translation by causing ribosomes to pause. In someembodiments, the use of lower abundance codon triplets can reducetranslational pausing due to a decrease in the pause time needed to loada charged tRNA into the ribosome translation machinery. Therefore, toincrease transcriptional and translational efficiency in bacteria (e.g.,where transcription and translation are concurrent, for example) or toincrease translational efficiency in eukaryotes (e.g., wheretranscription and translation are functionally separated), thenucleotide sequence of a nucleotide sequence of interest can be alteredto better suit the transcription and/or translational machinery of thehost and/or genetically modified microorganism. In certain embodiments,slowing the rate of translation by the use of lower abundance codons,which slow or pause the ribosome, can lead to higher yields of thedesired product due to an increase in correctly folded proteins and areduction in the formation of inclusion bodies.

Codons can be altered and optimized according to the preferred usage bya given organism by determining the codon distribution of the nucleotidesequence donor organism and comparing the distribution of codons to thedistribution of codons in the recipient or host organism. Techniquesdescribed herein (e.g., site directed mutagenesis and the like) can thenbe used to alter the codons accordingly. Comparisons of codon usage canbe done by hand, or using nucleic acid analysis software commerciallyavailable to the artisan.

Modification of the nucleotide sequence of an ORF also can be used tocorrect codon triplet sequences that have diverged in differentorganisms. For example, certain yeast (e.g., C. tropicalis and C.maltosa) use the amino acid triplet CUG (e.g., CTG in the DNA sequence)to encode serine. CUG typically encodes leucine in most organisms. Inorder to maintain the correct amino acid in the resultant polypeptide orprotein, the CUG codon must be altered to reflect the organism in whichthe nucleic acid reagent will be expressed. Thus, if an ORF from abacterial donor is to be expressed in either Candida yeast strainmentioned above, the heterologous nucleotide sequence must first bealtered or modified to the appropriate leucine codon. Therefore, in someembodiments, the nucleotide sequence of an ORF sometimes is altered ormodified to correct for differences that have occurred in the evolutionof the amino acid codon triplets between different organisms. In someembodiments, the nucleotide sequence can be left unchanged at aparticular amino acid codon, if the amino acid encoded is a conservativeor neutral change in amino acid when compared to the originally encodedamino acid.

In some embodiments, an activity can be altered by modifyingtranslational regulation signals, like a stop codon for example. A stopcodon at the end of an ORF sometimes is modified to another stop codon,such as an amber stop codon described above. In some embodiments, a stopcodon is introduced within an ORF, sometimes by insertion or mutation ofan existing codon. An ORF comprising a modified terminal stop codonand/or internal stop codon often is translated in a system comprising asuppressor tRNA that recognizes the stop codon. An ORF comprising a stopcodon sometimes is translated in a system comprising a suppressor tRNAthat incorporates an unnatural amino acid during translation of thetarget protein or target peptide. Methods for incorporating unnaturalamino acids into a target protein or peptide are known, which include,for example, processes utilizing a heterologous tRNA/synthetase pair,where the tRNA recognizes an amber stop codon and is loaded with anunnatural amino acid (e.g., World Wide Web URLiupac.org/news/prize/2003/wang.pdf).

Depending on the portion of a nucleic acid reagent (e.g., Promoter, 5′or 3′ UTR, ORI, ORF, and the like) chosen for alteration (e.g., bymutagenesis, introduction or deletion, for example) the modificationsdescribed above can alter a given activity by (i) increasing ordecreasing feedback inhibition mechanisms, (ii) increasing or decreasingpromoter initiation, (iii) increasing or decreasing translationinitiation, (iv) increasing or decreasing translational efficiency, (v)modifying localization of peptides or products expressed from nucleicacid reagents described herein, or (vi) increasing or decreasing thecopy number of a nucleotide sequence of interest, (vii) expression of ananti-sense RNA, RNAi, siRNA, ribozyme and the like. In some embodiments,alteration of a nucleic acid reagent or nucleotide sequence can alter aregion involved in feedback inhibition (e.g., 5′ UTR, promoter and thelike). A modification sometimes is made that can add or enhance bindingof a feedback regulator and sometimes a modification is made that canreduce, inhibit or eliminate binding of a feedback regulator.

In certain embodiments, alteration of a nucleic acid reagent ornucleotide sequence can alter sequences involved in transcriptioninitiation (e.g., promoters, 5′ UTR, and the like). A modificationsometimes can be made that can enhance or increase initiation from anendogenous or heterologous promoter element. A modification sometimescan be made that removes or disrupts sequences that increase or enhancetranscription initiation, resulting in a decrease or elimination oftranscription from an endogenous or heterologous promoter element.

In some embodiments, alteration of a nucleic acid reagent or nucleotidesequence can alter sequences involved in translational initiation ortranslational efficiency (e.g., 5′ UTR, 3′ UTR, codon triplets of higheror lower abundance, translational terminator sequences and the like, forexample). A modification sometimes can be made that can increase ordecrease translational initiation, modifying a ribosome binding site forexample. A modification sometimes can be made that can increase ordecrease translational efficiency. Removing or adding sequences thatform hairpins and changing codon triplets to a more or less preferredcodon are non-limiting examples of genetic modifications that can bemade to alter translation initiation and translation efficiency.

In certain embodiments, alteration of a nucleic acid reagent ornucleotide sequence can alter sequences involved in localization ofpeptides, proteins or other desired products (e.g., adipic acid, forexample). A modification sometimes can be made that can alter, add orremove sequences responsible for targeting a polypeptide, protein orproduct to an intracellular organelle, the periplasm, cellularmembranes, or extracellularly. Transport of a heterologous product to adifferent intracellular space or extracellularly sometimes can reduce oreliminate the formation of inclusion bodies (e.g., insoluble aggregatesof the desired product).

In some embodiments, alteration of a nucleic acid reagent or nucleotidesequence can alter sequences involved in increasing or decreasing thecopy number of a nucleotide sequence of interest. A modificationsometimes can be made that increases or decreases the number of copiesof an ORF stably integrated into the genome of an organism or on anepigenetic nucleic acid reagent. Non-limiting examples of alterationsthat can increase the number of copies of a sequence of interestinclude, adding copies of the sequence of interest by duplication ofregions in the genome (e.g., adding additional copies by recombinationor by causing gene amplification of the host genome, for example),cloning additional copies of a sequence onto a nucleic acid reagent, oraltering an ORI to increase the number of copies of an epigeneticnucleic acid reagent. Non-limiting examples of alterations that candecrease the number of copies of a sequence of interest include,removing copies of the sequence of interest by deletion or disruption ofregions in the genome, removing additional copies of the sequence fromepigenetic nucleic acid reagents, or altering an ORI to decrease thenumber of copies of an epigenetic nucleic acid reagent.

In certain embodiments, increasing or decreasing the expression of anucleotide sequence of interest can also be accomplished by altering,adding or removing sequences involved in the expression of an anti-senseRNA, RNAi, siRNA, ribozyme and the like. The methods described above canbe used to modify expression of anti-sense RNA, RNAi, siRNA, ribozymeand the like.

The methods and nucleic acid reagents described herein can be used togenerate genetically modified microorganisms with altered activities incellular processes involved in adipic acid synthesis. In someembodiments, an engineered microorganism described herein may comprise aheterologous polynucleotide encoding a polypeptide having 6-oxohexanoicacid dehydrogenase activity, and in certain embodiments, an engineeredmicroorganism described herein may comprise a heterologouspolynucleotide encoding a polypeptide having omega oxo fatty aciddehydrogenase activity. In some embodiments, an engineered microorganismdescribed herein may comprise a heterologous polynucleotide encoding apolypeptide having 6-hydroxyhexanoic acid dehydrogenase activity, and incertain embodiments, an engineered microorganism described herein maycomprise a heterologous polynucleotide encoding a polypeptide havingomega hydroxyl fatty acid dehydrogenase activity. In some embodiments,the heterologous polynucleotide can be from a bacterium. In someembodiments, the bacterium can be an Acinetobacter, Nocardia,Pseudomonas or Xanthobacter bacterium.

In certain embodiments, an engineered microorganism described herein maycomprise a heterologous polynucleotide encoding a polypeptide havinghexanoate synthase subunit A activity. In some embodiments, anengineered microorganism described herein may comprise a heterologouspolynucleotide encoding a polypeptide having hexanoate synthase subunitB activity. In certain embodiments, the heterologous polynucleotideindependently is selected from a fungus. In some embodiments, the funguscan be an Aspergillus fungus. In certain embodiments, the Aspergillusfungus is A. parasiticus.

In some embodiments, an engineered microorganism described herein maycomprise a heterologous polynucleotide encoding a polypeptide havingmonooxygenase activity. In certain embodiments, the heterologouspolynucleotide can be from a bacterium. In some embodiments, thebacterium can be a Bacillus bacterium. In certain embodiments, theBacillus bacterium is B. megaterium.

In some embodiments, an engineered microorganism described herein maycomprise a genetic modification that results in primary hexanoate usageby monooxygenase activity. In certain embodiments, the geneticmodification can reduce a polyketide synthase activity. In someembodiments, the engineered microorganism can be a eukaryote. In certainembodiments, the eukaryote can be a yeast. In some embodiments, theeukaryote may be a fungus. In certain embodiments, the yeast can be aCandida yeast. In some embodiments, the Candida yeast may be C.tropicalis. In certain embodiments, the fungus can be a Yarrowia fungus.In some embodiments the Yarrowia fungus may be Y. lipolytica. In certainembodiments, the fungus can be an Aspergillus fungus. In someembodiments, the Aspergillus fungus may be A. parasiticus or A.nidulans.

In certain embodiments, an engineered microorganism described herein maycomprise a genetic modification that reduces 6-hydroxyhexanoic acidconversion. In some embodiments, the genetic modification can reduce6-hydroxyhexanoic acid dehydrogenase activity. In certain embodiments,an engineered microorganism described herein may comprise a geneticmodification that reduces beta-oxidation activity. In some embodiments,the genetic modification can reduce a target activity described herein.

Engineered microorganisms that produce adipic acid, as described herein,can comprise an altered monooxygenase activity, in certain embodiments.In some embodiments, the engineered microorganism described herein maycomprise a genetic modification that alters the monooxygenase activity.In certain embodiments, the genetic modification can result insubstantial hexanoate usage by the monooxygenase activity. In someembodiments, the genetic modification can reduce a polyketide synthaseactivity. In certain embodiments, the engineered microorganism describedherein can comprise a heterologous polynucleotide encoding a polypeptidehaving monooxygenase activity. In certain embodiments, the heterologouspolynucleotide can be from a bacterium. In some embodiments, thebacterium can be a Bacillus bacterium. In certain embodiments, theBacillus bacterium is B. megaterium.

In some embodiments, the engineered microorganism described herein maycomprise an altered hexanoate synthase activity. In certain embodiments,the altered hexanoate synthase activity is an altered hexanoate synthasesubunit A activity, altered hexanoate synthase subunit B activity, oraltered hexanoate synthase subunit A activity and altered hexanoatesynthase subunit B activity. In some embodiments, the engineeredmicroorganism may comprise a genetic alteration that adds or increaseshexanoate synthase activity. In certain embodiments, the engineeredmicroorganism may comprise a heterologous polynucleotide encoding apolypeptide having hexanoate synthase activity. In some embodiments, theheterologous polynucleotide can be from a fungus. In certainembodiments, the fungus can be an Aspergillus fungus. In someembodiments, the Aspergillus fungus is A. parasiticus.

Engineered microorganisms that produce adipic acid, as described herein,can comprise an altered thioesterase activity, in certain embodiments.In some embodiments, the engineered microorganism may comprise a geneticmodification that adds or increases the thioesterase activity. Incertain embodiments, the engineered microorganism may comprise aheterologous polynucleotide encoding a polypeptide having thioesteraseactivity.

In some embodiments, the engineered microorganism with an alteredthioesterase activity may comprise an altered 6-oxohexanoic aciddehydrogenase activity, or an altered omega oxo fatty acid dehydrogenaseactivity. In certain embodiments, the engineered microorganism with analtered thioesterase activity may comprise a genetic modification thatadds or increases 6-oxohexanoic acid dehydrogenase activity, or agenetic modification that adds or increases omega oxo fatty aciddehydrogenase activity. In some embodiments, the engineeredmicroorganism may comprise a heterologous polynucleotide encoding apolypeptide having altered 6-oxohexanoic acid dehydrogenase activity,and in some embodiments, the engineered microorganism may comprise aheterologous polynucleotide encoding a polypeptide having altered omegaoxo fatty acid dehydrogenase activity. In certain embodiments, theheterologous polynucleotide can be from a bacterium. In someembodiments, the bacterium can be an Acinetobacter, Nocardia,Pseudomonas or Xanthobacter bacterium.

Engineered microorganisms that produce adipic acid, as described herein,can comprise an altered 6-hydroxyhexanoic acid dehydrogenase activity,in certain embodiments, and in some embodiments, can comprise an alteredomega hydroxyl fatty acid dehydrogenase activity. In certainembodiments, the engineered microorganism may comprise a geneticmodification that adds or increases the 6-hydroxyhexanoic aciddehydrogenase activity and in some embodiments the engineeredmicroorganism may comprise a genetic modification that adds or increasesthe omega hydroxyl fatty acid dehydrogenase activity. In certainembodiments, the engineered microorganism may comprise a heterologouspolynucleotide encoding a polypeptide having altered 6-hydroxyhexanoicacid dehydrogenase activity, and in some embodiments, the engineeredmicroorganism may comprise a heterologous polynucleotide encoding apolypeptide having altered omega hydroxyl fatty acid dehydrogenaseactivity. In some embodiments, the heterologous polynucleotide is from abacterium. In certain embodiments, the bacterium can be anAcinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium. In someembodiments, the engineered microorganism can be a eukaryote. In certainembodiments, the eukaryote can be a yeast. In some embodiments, theeukaryote may be a fungus. In certain embodiments, the yeast can be aCandida yeast. In some embodiments, the Candida yeast may be C.tropicalis. In certain embodiments, the fungus can be a Yarrowia fungus.In some embodiments the Yarrowia fungus may be Y. lipolytica. In certainembodiments, the fungus can be an Aspergillus fungus. In someembodiments, the Aspergillus fungus may be A. parasiticus or A.nidulans.

In some embodiments, an engineered microorganism as described above maycomprise a genetic modification that reduces 6-hydroxyhexanoic acidconversion. In certain embodiments, the genetic modification can reduce6-hydroxyhexanoic acid dehydrogenase activity. In some embodiments thegenetic may reduce beta-oxidation activity. In certain embodiments, thegenetic modification may reduce a target activity described herein.

Engineered microorganisms can be prepared by altering, introducing orremoving nucleotide sequences in the host genome or in stably maintainedepigenetic nucleic acid reagents, as noted above. The nucleic acidreagents use to alter, introduce or remove nucleotide sequences in thehost genome or epigenetic nucleic acids can be prepared using themethods described herein or available to the artisan.

Nucleic acid sequences having a desired activity can be isolated fromcells of a suitable organism using lysis and nucleic acid purificationprocedures described in a known reference manual (e.g., Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular Cloning: a LaboratoryManual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) orusing commercially available cell lysis and DNA purification reagentsand kits. In some embodiments, nucleic acids used to engineermicroorganisms can be provided for conducting methods described hereinafter processing of the organism containing the nucleic acid. Forexample, the nucleic acid of interest may be extracted, isolated,purified or amplified from a sample (e.g., from an organism of interestor culture containing a plurality of organisms of interest, like yeastor bacteria for example). The term “isolated” as used herein refers tonucleic acid removed from its original environment (e.g., the naturalenvironment if it is naturally occurring, or a host cell if expressedexogenously), and thus is altered “by the hand of man” from its originalenvironment. An isolated nucleic acid generally is provided with fewernon-nucleic acid components (e.g., protein, lipid) than the amount ofcomponents present in a source sample. A composition comprising isolatedsample nucleic acid can be substantially isolated (e.g., about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free ofnon-nucleic acid components). The term “purified” as used herein refersto sample nucleic acid provided that contains fewer nucleic acid speciesthan in the sample source from which the sample nucleic acid is derived.A composition comprising sample nucleic acid may be substantiallypurified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or greater than 99% free of other nucleic acid species). The term“amplified” as used herein refers to subjecting nucleic acid of a cell,organism or sample to a process that linearly or exponentially generatesamplicon nucleic acids having the same or substantially the samenucleotide sequence as the nucleotide sequence of the nucleic acid inthe sample, or portion thereof. As noted above, the nucleic acids usedto prepare nucleic acid reagents as described herein can be subjected tofragmentation or cleavage.

Amplification of nucleic acids is sometimes necessary when dealing withorganisms that are difficult to culture. Where amplification may bedesired, any suitable amplification technique can be utilized.Non-limiting examples of methods for amplification of polynucleotidesinclude, polymerase chain reaction (PCR); ligation amplification (orligase chain reaction (LCR)); amplification methods based on the use ofQ-beta replicase or template-dependent polymerase (see US PatentPublication Number US20050287592); helicase-dependant isothermalamplification (Vincent et al., “Helicase-dependent isothermal DNAamplification”. EMBO reports 5 (8): 795-800 (2004)); strand displacementamplification (SDA); thermophilic SDA nucleic acid sequence basedamplification (3SR or NASBA) and transcription-associated amplification(TAA). Non-limiting examples of PCR amplification methods includestandard PCR, AFLP-PCR, Allele-specific PCR, Alu-PCR, Asymmetric PCR,Colony PCR, Hot start PCR, Inverse PCR (IPCR), In situ PCR (ISH),Intersequence-specific PCR (ISSR-PCR), Long PCR, Multiplex PCR, NestedPCR, Quantitative PCR, Reverse Transcriptase PCR(RT-PCR), Real Time PCR,Single cell PCR, Solid phase PCR, combinations thereof, and the like.Reagents and hardware for conducting PCR are commercially available.

Protocols for conducting the various type of PCR listed above arereadily available to the artisan. PCR conditions can be dependent uponprimer sequences, target abundance, and the desired amount ofamplification, and therefore, one of skill in the art may choose from anumber of PCR protocols available (see, e.g., U.S. Pat. Nos. 4,683,195and 4,683,202; and PCR Protocols: A Guide to Methods and Applications,Innis et al., eds, 1990. PCR often is carried out as an automatedprocess with a thermostable enzyme. In this process, the temperature ofthe reaction mixture is cycled through a denaturing region, aprimer-annealing region, and an extension reaction region automatically.Machines specifically adapted for this purpose are commerciallyavailable. A non-limiting example of a PCR protocol that may be suitablefor embodiments described herein is, treating the sample at 95° C. for 5minutes; repeating forty-five cycles of 95° C. for 1 minute, 59° C. for1 minute, 10 seconds, and 72° C. for 1 minute 30 seconds; and thentreating the sample at 72° C. for 5 minutes. Additional PCR protocolsare described in the example section. Multiple cycles frequently areperformed using a commercially available thermal cycler. Suitableisothermal amplification processes known and selected by the person ofordinary skill in the art also may be applied, in certain embodiments.In some embodiments, nucleic acids encoding polypeptides with a desiredactivity can be isolated by amplifying the desired sequence from anorganism having the desired activity using oligonucleotides or primersdesigned based on sequences described herein.

Amplified, isolated and/or purified nucleic acids can be cloned into therecombinant DNA vectors described in Figures herein or into suitablecommercially available recombinant DNA vectors. Cloning of nucleic acidsequences of interest into recombinant DNA vectors can facilitatefurther manipulations of the nucleic acids for preparation of nucleicacid reagents, (e.g., alteration of nucleotide sequences by mutagenesis,homologous recombination, amplification and the like, for example).Standard cloning procedures (e.g., enzymic digestion, ligation, and thelike) are known (e.g., described in Maniatis, T., E. F. Fritsch and J.Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

In some embodiments, nucleic acid sequences prepared by isolation oramplification can be used, without any further modification, to add anactivity to a microorganism and thereby create a genetically modified orengineered microorganism. In certain embodiments, nucleic acid sequencesprepared by isolation or amplification can be genetically modified toalter (e.g., increase or decrease, for example) a desired activity. Insome embodiments, nucleic acids, used to add an activity to an organism,sometimes are genetically modified to optimize the heterologouspolynucleotide sequence encoding the desired activity (e.g., polypeptideor protein, for example). The term “optimize” as used herein can referto alteration to increase or enhance expression by preferred codonusage. The term optimize can also refer to modifications to the aminoacid sequence to increase the activity of a polypeptide or protein, suchthat the activity exhibits a higher catalytic activity as compared tothe “natural” version of the polypeptide or protein.

Nucleic acid sequences of interest can be genetically modified usingmethods known in the art. Mutagenesis techniques are particularly usefulfor small scale (e.g., 1, 2, 5, 10 or more nucleotides) or large scale(e.g., 50, 100, 150, 200, 500, or more nucleotides) geneticmodification. Mutagenesis allows the artisan to alter the geneticinformation of an organism in a stable manner, either naturally (e.g.,isolation using selection and screening) or experimentally by the use ofchemicals, radiation or inaccurate DNA replication (e.g., PCRmutagenesis). In some embodiments, genetic modification can be performedby whole scale synthetic synthesis of nucleic acids, using a nativenucleotide sequence as the reference sequence, and modifying nucleotidesthat can result in the desired alteration of activity. Mutagenesismethods sometimes are specific or targeted to specific regions ornucleotides (e.g., site-directed mutagenesis, PCR-based site-directedmutagenesis, and in vitro mutagenesis techniques such as transplacementand in vivo oligonucleotide site-directed mutagenesis, for example).Mutagenesis methods sometimes are non-specific or random with respect tothe placement of genetic modifications (e.g., chemical mutagenesis,insertion element (e.g., insertion or transposon elements) andinaccurate PCR based methods, for example).

Site directed mutagenesis is a procedure in which a specific nucleotideor specific nucleotides in a DNA molecule are mutated or altered. Sitedirected mutagenesis typically is performed using a nucleic acidsequence of interest cloned into a circular plasmid vector.Site-directed mutagenesis requires that the wild type sequence be knownand used a platform for the genetic alteration. Site-directedmutagenesis sometimes is referred to as oligonucleotide-directedmutagenesis because the technique can be performed usingoligonucleotides which have the desired genetic modificationincorporated into the complement a nucleotide sequence of interest. Thewild type sequence and the altered nucleotide are allowed to hybridizeand the hybridized nucleic acids are extended and replicated using a DNApolymerase. The double stranded nucleic acids are introduced into a host(e.g., E. coli, for example) and further rounds of replication arecarried out in vivo. The transformed cells carrying the mutated nucleicacid sequence are then selected and/or screened for those cells carryingthe correctly mutagenized sequence. Cassette mutagenesis and PCR-basedsite-directed mutagenesis are further modifications of the site-directedmutagenesis technique. Site-directed mutagenesis can also be performedin vivo (e.g., transplacement “pop-in pop-out”, In vivo site-directedmutagenesis with synthetic oligonucleotides and the like, for example).

PCR-based mutagenesis can be performed using PCR with oligonucleotideprimers that contain the desired mutation or mutations. The techniquefunctions in a manner similar to standard site-directed mutagenesis,with the exception that a thermocycler and PCR conditions are used toreplace replication and selection of the clones in a microorganism host.As PCR-based mutagenesis also uses a circular plasmid vector, theamplified fragment (e.g., linear nucleic acid molecule) containing theincorporated genetic modifications can be separated from the plasmidcontaining the template sequence after a sufficient number of rounds ofthermocycler amplification, using standard electrophorectic procedures.A modification of this method uses linear amplification methods and apair of mutagenic primers that amplify the entire plasmid. The proceduretakes advantage of the E. coli Dam methylase system which causes DNAreplicated in vivo to be sensitive to the restriction endonucleasesDpnl. PCR synthesized DNA is not methylated and is therefore resistantto Dpnl. This approach allows the template plasmid to be digested,leaving the genetically modified, PCR synthesized plasmids to beisolated and transformed into a host bacteria for DNA repair andreplication, thereby facilitating subsequent cloning and identificationsteps. A certain amount of randomness can be added to PCR-based siteddirected mutagenesis by using partially degenerate primers.

Recombination sometimes can be used as a tool for mutagenesis.Homologous recombination allows the artisan to specifically targetregions of known sequence for insertion of heterologous nucleotidesequences using the host organisms natural DNA replication and repairenzymes. Homologous recombination methods sometimes are referred to as“pop in pop out” mutagenesis, transplacement, knock out mutagenesis orknock in mutagenesis. Integration of a nucleic acid sequence into a hostgenome is a single cross over event, which inserts the entire nucleicacid reagent (e.g., pop in). A second cross over event excises all but aportion of the nucleic acid reagent, leaving behind a heterologoussequence, often referred to as a “footprint” (e.g., pop out).Mutagenesis by insertion (e.g., knock in) or by double recombinationleaving behind a disrupting heterologous nucleic acid (e.g., knock out)both server to disrupt or “knock out” the function of the gene ornucleic acid sequence in which insertion occurs. By combining selectablemarkers and/or auxotrophic markers with nucleic acid reagents designedto provide the appropriate nucleic acid target sequences, the artisancan target a selectable nucleic acid reagent to a specific region, andthen select for recombination events that “pop out” a portion of theinserted (e.g., “pop in”) nucleic acid reagent.

Such methods take advantage of nucleic acid reagents that have beenspecifically designed with known target nucleic acid sequences at ornear a nucleic acid or genomic region of interest. Popping out typicallyleaves a “foot print” of left over sequences that remain after therecombination event. The left over sequence can disrupt a gene andthereby reduce or eliminate expression of that gene. In someembodiments, the method can be used to insert sequences, upstream ordownstream of genes that can result in an enhancement or reduction inexpression of the gene. In certain embodiments, new genes can beintroduced into the genome of a host organism using similarrecombination or “pop in” methods. An example of a yeast recombinationsystem using the ura3 gene and 5-FOA were described briefly above andfurther detail is presented herein.

A method for modification is described in Alani et al., “A method forgene disruption that allows repeated use of URA3 selection in theconstruction of multiply disrupted yeast strains”, Genetics116(4):541-545 August 1987. The original method uses a Ura3 cassettewith 1000 base pairs (bp) of the same nucleotide sequence cloned in thesame orientation on either side of the URA3 cassette. Targetingsequences of about 50 bp are added to each side of the construct. Thedouble stranded targeting sequences are complementary to sequences inthe genome of the host organism. The targeting sequences allowsite-specific recombination in a region of interest. The modification ofthe original technique replaces the two 1000 bp sequence direct repeatswith two 200 bp direct repeats. The modified method also uses 50 bptargeting sequences. The modification reduces or eliminatesrecombination of a second knock out into the 1000 bp repeat left behindin a first mutagenesis, therefore allowing multiply knocked out yeast.Additionally, the 200 bp sequences used herein are uniquely designed,self-assembling sequences that leave behind identifiable footprints. Thetechnique used to design the sequences incorporate design features suchas low identity to the yeast genome, and low identity to each other.Therefore a library of the self-assembling sequences can be generated toallow multiple knockouts in the same organism, while reducing oreliminating the potential for integration into a previous knockout.

As noted above, the URA3 cassette makes use of the toxicity of 5-FOA inyeast carrying a functional URA3 gene. Uracil synthesis deficient yeastare transformed with the modified URA3 cassette, using standard yeasttransformation protocols, and the transformed cells are plated onminimal media minus uracil. In some embodiments, PCR can be used toverify correct insertion into the region of interest in the host genome,and certain embodiments the PCR step can be omitted. Inclusion of thePCR step can reduce the number of transformants that need to be counterselected to “pop out” the URA3 cassette. The transformants (e.g., all orthe ones determined to be correct by PCR, for example) can then becounter-selected on media containing 5-FOA, which will select forrecombination out (e.g., popping out) of the URA3 cassette, thusrendering the yeast ura3 deficient again, and resistant to 5-FOAtoxicity. Targeting sequences used to direct recombination events tospecific regions are presented herein. A modification of the methoddescribed above can be used to integrate genes in to the chromosome,where after recombination a functional gene is left in the chromosomenext to the 200 bp footprint.

In some embodiments, other auxotrophic or dominant selection markers canbe used in place of URA3 (e.g., an auxotrophic selectable marker), withthe appropriate change in selection media and selection agents.Auxotrophic selectable markers are used in strains deficient forsynthesis of a required biological molecule (e.g., amino acid ornucleoside, for example). Non-limiting examples of additionalauxotrophic markers include; HIS3, TRP1, LEU2, LEU2-d, and LYS2. Certainauxotrophic markers (e.g., URA3 and LYS2) allow counter selection toselect for the second recombination event that pops out all but one ofthe direct repeats of the recombination construct. HIS3 encodes anactivity involved in histidine synthesis. TRP1 encodes an activityinvolved in tryptophan synthesis. LEU2 encodes an activity involved inleucine synthesis. LEU2-d is a low expression version of LEU2 thatselects for increased copy number (e.g., gene or plasmid copy number,for example) to allow survival on minimal media without leucine. LYS2encodes an activity involved in lysine synthesis, and allows counterselection for recombination out of the LYS2 gene using alpha-aminoadipate (α-amino adipate).

Dominant selectable markers are useful because they also allowindustrial and/or prototrophic strains to be used for geneticmanipulations. Additionally, dominant selectable markers provide theadvantage that rich medium can be used for plating and culture growth,and thus growth rates are markedly increased. Non-limiting examples ofdominant selectable markers include; Tn903 kan^(r), Cm^(r), Hyg^(r),CUP1, and DHFR. Tn903 kan^(r) encodes an activity involved in kanamycinantibiotic resistance (e.g., typically neomycin phosphotransferase II orNPTII, for example). Cm^(r) encodes an activity involved inchloramphenicol antibiotic resistance (e.g., typically chloramphenicolacetyl transferase or CAT, for example). Hyg^(r) encodes an activityinvolved in hygromycin resistance by phosphorylation of hygromycin B(e.g., hygromycin phosphotransferase, or HPT). CUP1 encodes an activityinvolved in resistance to heavy metal (e.g., copper, for example)toxicity. DHFR encodes a dihydrofolate reductase activity which confersresistance to methotrexate and sulfanilamide compounds.

In contrast to site-directed or specific mutagenesis, random mutagenesisdoes not require any sequence information and can be accomplished by anumber of widely different methods. Random mutagenesis often is used tocreate mutant libraries that can be used to screen for the desiredgenotype or phenotype. Non-limiting examples of random mutagenesisinclude; chemical mutagenesis, UV-induced mutagenesis, insertion elementor transposon-mediated mutagenesis, DNA shuffling, error-prone PCRmutagenesis, and the like.

Chemical mutagenesis often involves chemicals like ethylmethanesulfonate (EMS), nitrous acid, mitomycin C,N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1,2,7,8-diepoxyoctane(DEO), methyl methane sulfonate (MMS),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide(4-NQO),2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)-acridinedihydrochloride(ICR-170), 2-amino purine (2AP), and hydroxylamine (HA), provided hereinas non-limiting examples. These chemicals can cause base-pairsubstitutions, frameshift mutations, deletions, transversion mutations,transition mutations, incorrect replication, and the like. In someembodiments, the mutagenesis can be carried out in vivo. Sometimes themutagenic process involves the use of the host organisms DNA replicationand repair mechanisms to incorporate and replicate the mutagenized baseor bases.

Another type of chemical mutagenesis involves the use of base-analogs.The use of base-analogs cause incorrect base pairing which in thefollowing round of replication is corrected to a mismatched nucleotidewhen compared to the starting sequence. Base analog mutagenesisintroduces a small amount of non-randomness to random mutagenesis,because specific base analogs can be chose which can be incorporated atcertain nucleotides in the starting sequence. Correction of themispairing typically yields a known substitution. For example,Bromo-deoxyuridine (BrdU) can be incorporated into DNA and replaces T inthe sequence. The host DNA repair and replication machinery can sometimecorrect the defect, but sometimes will mispair the BrdU with a G. Thenext round of replication then causes a G-C transversion from theoriginal A-T in the native sequence.

Ultra violet (UV) induced mutagenesis is caused by the formation ofthymidine dimers when UV light irradiates chemical bonds between twoadjacent thymine residues. Excision repair mechanism of the hostorganism correct the lesion in the DNA, but occasionally the lesion isincorrectly repaired typically resulting in a C to T transition.

Insertion element or transposon-mediated mutagenesis makes use ofnaturally occurring or modified naturally occurring mobile geneticelements. Transposons often encode accessory activities in addition tothe activities necessary for transposition (e.g., movement using atransposase activity, for example). In many examples, transposonaccessory activities are antibiotic resistance markers (e.g., see Tn903kan^(r) described above, for example). Insertion elements typically onlyencode the activities necessary for movement of the nucleic acidsequence. Insertion element and transposon mediated mutagenesis oftencan occur randomly, however specific target sequences are known for sometransposons. Mobile genetic elements like IS elements or Transposons(Tn) often have inverted repeats, direct repeats or both inverted anddirect repeats flanking the region coding for the transposition genes.Recombination events catalyzed by the transposase cause the element toremove itself from the genome and move to a new location, leaving behinda portion of an inverted or direct repeat. Classic examples oftransposons are the “mobile genetic elements” discovered in maize.Transposon mutagenesis kits are commercially available which aredesigned to leave behind a 5 codon insert (e.g., Mutation GenerationSystem kit, Finnzymes, World Wide Web URL finnzymes.us, for example).This allows the artisan to identify the insertion site, without fullydisrupting the function of most genes.

DNA shuffling is a method which uses DNA fragments from members of amutant library and reshuffles the fragments randomly to generate newmutant sequence combinations. The fragments are typically generatedusing DNasel, followed by random annealing and re-joining using selfpriming PCR. The DNA overhanging ends, from annealing of randomfragments, provide “primer” sequences for the PCR process. Shuffling canbe applied to libraries generated by any of the above mutagenesismethods.

Error prone PCR and its derivative rolling circle error prone PCR usesincreased magnesium and manganese concentrations in conjunction withlimiting amounts of one or two nucleotides to reduce the fidelity of theTaq polymerase. The error rate can be as high as 2% under appropriateconditions, when the resultant mutant sequence is compared to the wildtype starting sequence. After amplification, the library of mutantcoding sequences must be cloned into a suitable plasmid. Although pointmutations are the most common types of mutation in error prone PCR,deletions and frameshift mutations are also possible. There are a numberof commercial error-prone PCR kits available, including those fromStratagene and Clontech (e.g., World Wide Web URL strategene.com andWorld Wide Web URL clontech.com, respectively, for example). Rollingcircle error-prone PCR is a variant of error-prone PCR in whichwild-type sequence is first cloned into a plasmid, then the wholeplasmid is amplified under error-prone conditions.

As noted above, organisms with altered activities can also be isolatedusing genetic selection and screening of organisms challenged onselective media or by identifying naturally occurring variants fromunique environments. For example, 2-Deoxy-D-glucose is a toxic glucoseanalog. Growth of yeast on this substance yields mutants that areglucose-deregulated. A number of mutants have been isolated using2-Deoxy-D-glucose including transport mutants, and mutants that fermentglucose and galactose simultaneously instead of glucose first thengalactose when glucose is depleted. Similar techniques have been used toisolate mutant microorganisms that can metabolize plastics (e.g., fromlandfills), petrochemicals (e.g., from oil spills), and the like, eitherin a laboratory setting or from unique environments.

Similar methods can be used to isolate naturally occurring mutations ina desired activity when the activity exists at a relatively low ornearly undetectable level in the organism of choice, in someembodiments. The method generally consists of growing the organism to aspecific density in liquid culture, concentrating the cells, and platingthe cells on various concentrations of the substance to which anincrease in metabolic activity is desired. The cells are incubated at amoderate growth temperature, for 5 to 10 days. To enhance the selectionprocess, the plates can be stored for another 5 to 10 days at a lowtemperature. The low temperature sometimes can allow strains that havegained or increased an activity to continue growing while other strainsare inhibited for growth at the low temperature. Following the initialselection and secondary growth at low temperature, the plates can bereplica plated on higher or lower concentrations of the selectionsubstance to further select for the desired activity.

A native, heterologous or mutagenized polynucleotide can be introducedinto a nucleic acid reagent for introduction into a host organism,thereby generating an engineered microorganism. Standard recombinant DNAtechniques (restriction enzyme digests, ligation, and the like) can beused by the artisan to combine the mutagenized nucleic acid of interestinto a suitable nucleic acid reagent capable of (i) being stablymaintained by selection in the host organism, or (ii) being integratinginto the genome of the host organism. As noted above, sometimes nucleicacid reagents comprise two replication origins to allow the same nucleicacid reagent to be manipulated in bacterial before final introduction ofthe final product into the host organism (e.g., yeast or fungus forexample). Standard molecular biology and recombinant DNA methods areknown (e.g., described in Maniatis, T., E. F. Fritsch and J. Sambrook(1982) Molecular Cloning: a Laboratory Manual; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

Nucleic acid reagents can be introduced into microorganisms usingvarious techniques. Non-limiting examples of methods used to introduceheterologous nucleic acids into various organisms include;transformation, transfection, transduction, electroporation,ultrasound-mediated transformation, particle bombardment and the like.In some instances the addition of carrier molecules (e.g.,bis-benzimdazolyl compounds, for example, see U.S. Pat. No. 5,595,899)can increase the uptake of DNA in cells typically though to be difficultto transform by conventional methods. Conventional methods oftransformation are known (e.g., described in Maniatis, T., E. F. Fritschand J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Culture, Production and Process Methods

Engineered microorganisms often are cultured under conditions thatoptimize yield of a target molecule (e.g., six-carbon target molecule).Non-limiting examples of such target molecules are adipic acid and6-hydroxyhexanoic acid. Culture conditions often optimize activity ofone or more of the following activities: 6-oxohexanoic aciddehydrogenase activity, omega oxo fatty acid dehydrogenase activity,6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity, hexanoate synthase, monooxygenase, monooxygenasereductase, fatty alcohol oxidase, acyl-CoA ligase, acyl-CoA oxidase,enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and/or acetyl-CoAC-acyltransferase activities. In general, non-limiting examples ofconditions that may be optimized include the type and amount of carbonsource, the type and amount of nitrogen source, the carbon-to-nitrogenratio, the oxygen level, growth temperature, pH, length of the biomassproduction phase, length of target product accumulation phase, and timeof cell harvest.

Culture media generally contain a suitable carbon source. Carbon sourcesuseful for culturing microorganisms and/or fermentation processessometimes are referred to as feedstocks. The term “feedstock” as usedherein refers to a composition containing a carbon source that isprovided to an organism, which is used by the organism to produce energyand metabolic products useful for growth. A feedstock may be a naturalsubstance, a “man-made substance,” a purified or isolated substance, amixture of purified substances, a mixture of unpurified substances orcombinations thereof. A feedstock often is prepared by and/or providedto an organism by a person, and a feedstock often is formulated prior toadministration to the organism. A carbon source may include, but is notlimited to including, one or more of the following substances:monosaccharides (e.g., also referred to as “saccharides,” which include6-carbon sugars (e.g., glucose, fructose), 5-carbon sugars (e.g., xyloseand other pentoses) and the like), disaccharides (e.g., lactose,sucrose), oligosaccharides (e.g., glycans, homopolymers of amonosaccharide), polysaccharides (e.g., starch, cellulose,heteropolymers of monosaccharides or mixtures thereof), sugar alcohols(e.g., glycerol), and renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Carbon sources alsocan be selected from one or more of the following non-limiting examples:paraffin (e.g., saturated paraffin, unsaturated paraffin, substitutedparaffin, linear paraffin, branched paraffin, or combinations thereof);alkanes (e.g., hexane), alkenes or alkynes, each of which may be linear,branched, saturated, unsaturated, substituted or combinations thereof(described in greater detail below); linear or branched alcohols (e.g.,hexanol); fatty acids (e.g., about 1 carbon to about 60 carbons,including free fatty acids, soap stock, for example); esters of fattyacids; monoglycerides; diglycerides; triglycerides, phospholipids.Non-limiting commercial sources of products for preparing feedstocksinclude plants or plant products (e.g., vegetable oils (e.g., almondoil, canola oil, cocoa butter, coconut oil, corn oil, cottonseed oil,flaxseed oil, grape seed oil, illipe, olive oil, palm oil, palm olein,palm kernel oil, safflower oil, peanut oil, soybean oil, sesame oil,shea nut oil, sunflower oil walnut oil, the like and combinationsthereof) and animal fats (e.g., beef tallow, butterfat, lard, cod liveroil). A carbon source may include a petroleum product and/or a petroleumdistillate (e.g., diesel, fuel oils, gasoline, kerosene, paraffin wax,paraffin oil, petrochemicals).

The term “paraffin” as used herein refers to the common name for alkanehydrocarbons, independent of the source (e.g., plant derived, petroleumderived, chemically synthesized, fermented by a microorganism), orcarbon chain length. A carbon source sometimes comprises a paraffin, andin some embodiments, a paraffin is predominant in a carbon source (e.g.,about 75%, 80%, 85%, 90% or 95% paraffin). A paraffin sometimes issaturated (e.g., fully saturated), sometimes includes one or moreunsaturations (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 unsaturations)and sometimes is substituted with one or more non-hydrogen substituents.Non-limiting examples of non-hydrogen substituents include halo, acetyl,═O, ═N—CN, ═N—OR, ═NR, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂,NRCOOR, NRCOR, CN, COOR, CONR₂, OOCR, COR, and NO₂, where each R isindependently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl, and each R isoptionally substituted with halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′₂,SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′,CONR′₂, OOCR′, COR′, and NO₂, where each R′ is independently H, C1-C8alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl orC5-C10 heteroaryl.

In some embodiments a feedstock is selected according to the genotypeand/or phenotype of the engineered microorganism to be cultured. Forexample, a feedstock rich in 12-carbon fatty acids, 12-carbondicarboxylic acids or 12-carbon paraffins, or a mixture of 10-carbon and12-carbon compounds can be useful for culturing yeast strains harboringan alteration that partially blocks beta oxidation by disrupting POX4activity, as described herein. Non-limiting examples of carbon sourceshaving 10 and/or 12 carbons include fats (e.g., coconut oil, palm kerneloil), paraffins (e.g., alkanes, alkenes, or alkynes) having 10 or 12carbons, (e.g., decane, dodecane (also referred to as adakane 12,bihexyl, dihexyl and duodecane), alkene and alkyne derivatives), fattyacids (decanoic acid, dodecanoic acid), fatty alcohols (decanol,dodecanol), the like, non-toxic substituted derivatives or combinationsthereof.

A carbon source sometimes comprises an alkyl, alkenyl or alkynylcompound or molecule (e.g., a compound that includes an alkyl, alkenylor alkynyl moiety (e.g., alkane, alkene, alkyne)). In certainembodiments, an alkyl, alkenyl or alkynyl molecule, or combinationthereof, is predominant in a carbon source (e.g., about 75%, 80%, 85%,90% or 95% of such molecules). As used herein, the terms “alkyl,”“alkenyl” and “alkynyl” include straight-chain (referred to herein as“linear”), branched-chain (referred to herein as “non-linear”), cyclicmonovalent hydrocarbyl radicals, and combinations of these, whichcontain only C and H atoms when they are unsubstituted. Non-limitingexamples of alkyl moieties include methyl, ethyl, isobutyl, cyclohexyl,cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. An alkyl thatcontains only C and H atoms and is unsubstituted sometimes is referredto as “saturated.” An alkenyl or alkynyl generally is “unsaturated” asit contains one or more double bonds or triple bonds, respectively. Analkenyl can include any number of double bonds, such as 1, 2, 3, 4 or 5double bonds, for example. An alkynyl can include any number of triplebonds, such as 1, 2, 3, 4 or 5 triple bonds, for example. Alkyl, alkenyland alkynyl molecules sometimes contain between about 2 to about 60carbon atoms (C). For example, an alkyl, alkenyl and alkynyl moleculecan include about 1 carbon atom, about 2 carbon atoms, about 3 carbonatoms, about 4 carbon atoms, about 5 carbon atoms, about 6 carbon atoms,about 7 carbon atoms, about 8 carbon atoms, about 9 carbon atoms, about10 carbon atoms, about 12 carbon atoms, about 14 carbon atoms, about 16carbon atoms, about 18 carbon atoms, about 20 carbon atoms, about 22carbon atoms, about 24 carbon atoms, about 26 carbon atoms, about 28carbon atoms, about 30 carbon atoms, about 32 carbon atoms, about 34carbon atoms, about 36 carbon atoms, about 38 carbon atoms, about 40carbon atoms, about 42 carbon atoms, about 44 carbon atoms, about 46carbon atoms, about 48 carbon atoms, about 50 carbon atoms, about 52carbon atoms, about 54 carbon atoms, about 56 carbon atoms, about 58carbon atoms or about 60 carbon atoms. In some embodiments, paraffinscan have a mean number of carbon atoms of between about 8 to about 18carbon atoms (e.g., about 8 carbon atoms, about 9 carbon atoms, about 10carbon atoms, about 11 carbon atoms, about 12 carbon atoms, about 13carbon atoms, about 14 carbon atoms, about 15 carbon atoms, about 16carbon atoms, about 17 carbon atoms and about 18 carbon atoms). A singlegroup can include more than one type of multiple bond, or more than onemultiple bond. Such groups are included within the definition of theterm “alkenyl” when they contain at least one carbon-carbon double bond,and are included within the term “alkynyl” when they contain at leastone carbon-carbon triple bond. Alkyl, alkenyl and alkynyl moleculesinclude molecules that comprise an alkyl, alkenyl and/or alkynyl moiety,and include molecules that consist of an alkyl, alkenyl or alkynylmoiety (i.e., alkane, alkene and alkyne molecules).

Alkyl, alkenyl and alkynyl substituents sometimes contain 1-20 C (alkyl)or 2-20 C (alkenyl or alkynyl). They can contain about 8-14 C or about10-14 C in some embodiments. A single group can include more than onetype of multiple bond, or more than one multiple bond. Such groups areincluded within the definition of the term “alkenyl” when they containat least one carbon-carbon double bond, and are included within the term“alkynyl” when they contain at least one carbon-carbon triple bond.

Alkyl, alkenyl and alkynyl groups or compounds sometimes are substitutedto the extent that such substitution can be synthesized and can exist.Typical substituents include, but are not limited to, halo, acetyl, ═O,═N—CN, ═N—OR, ═NR, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR,NRCOR, CN, COOR, CONR₂, OOCR, COR, and NO₂, where each R isindependently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8heteroalkynyl, C6-C11 aryl, or C5-C11 heteroaryl, and each R isoptionally substituted with halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′₂,SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′,CONR′₂, OOCR′, COR′, and NO₂, where each R′ is independently H, C1-C8alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl orC5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups can also besubstituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10heteroaryl, each of which can be substituted by the substituents thatare appropriate for the particular group.

“Acetylene” or “acetyl” substituents are 2-10 C alkynyl groups that areoptionally substituted, and are of the formula —C≡C—Ri, where Ri is H orC1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl,C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl,and each Ri group is optionally substituted with one or moresubstituents selected from halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′2,SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′,CONR′₂, OOCR′, COR′, and NO₂, where each R′ is independently H, C1-C6alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C10 aryl,C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each ofwhich is optionally substituted with one or more groups selected fromhalo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl,hydroxy, amino, and ═O; and where two R′ can be linked to form a 3-7membered ring optionally containing up to three heteroatoms selectedfrom N, O and S. In some embodiments, Ri of —C≡C—Ri is H or Me.

A carbon source sometimes comprises a heteroalkyl, heteroalkenyl and/orheteroalkynyl molecule or compound (e.g., comprises heteroalkyl,heteroalkenyl and/or heteroalkynyl moiety (e.g., heteroalkane,heteroalkene or heteroalkyne)). “Heteroalkyl”, “heteroalkenyl”, and“heteroalkynyl” and the like are defined similarly to the correspondinghydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the ‘hetero’ termsrefer to groups that contain one to three O, S or N heteroatoms orcombinations thereof within the backbone; thus at least one carbon atomof a corresponding alkyl, alkenyl, or alkynyl group is replaced by oneof the specified heteroatoms to form a heteroalkyl, heteroalkenyl, orheteroalkynyl group. The typical and sizes for heteroforms of alkyl,alkenyl and alkynyl groups are generally the same as for thecorresponding hydrocarbyl groups, and the substituents that may bepresent on the heteroforms are the same as those described above for thehydrocarbyl groups. For reasons of chemical stability, it is alsounderstood that, unless otherwise specified, such groups do not includemore than two contiguous heteroatoms except where an oxo group ispresent on N or S as in a nitro or sulfonyl group.

The term “alkyl” as used herein includes cycloalkyl and cycloalkylalkylgroups and compounds, the term “cycloalkyl” may be used herein todescribe a carbocyclic non-aromatic compound or group that is connectedvia a ring carbon atom, and “cycloalkylalkyl” may be used to describe acarbocyclic non-aromatic compound or group that is connected to amolecule through an alkyl linker. Similarly, “heterocyclyl” may be usedto describe a non-aromatic cyclic group that contains at least oneheteroatom as a ring member and that is connected to the molecule via aring atom, which may be C or N; and “heterocyclylalkyl” may be used todescribe such a group that is connected to another molecule through alinker. The sizes and substituents that are suitable for the cycloalkyl,cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the sameas those described above for alkyl groups. As used herein, these termsalso include rings that contain a double bond or two, as long as thering is not aromatic.

A carbon source sometimes comprises an acyl compound or moiety (e.g.,compound comprising an acyl moiety). As used herein, “acyl” encompassesgroups comprising an alkyl, alkenyl, alkynyl, aryl or arylalkyl radicalattached at one of the two available valence positions of a carbonylcarbon atom, and heteroacyl refers to the corresponding groups where atleast one carbon other than the carbonyl carbon has been replaced by aheteroatom chosen from N, O and S. Thus heteroacyl includes, forexample, —C(═O)OR and —C(═O)NR₂ as well as —C(═O)-heteroaryl.

Acyl and heteroacyl groups are bonded to any group or molecule to whichthey are attached through the open valence of the carbonyl carbon atom.Typically, they are C1-C8 acyl groups, which include formyl, acetyl,pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, which includemethoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups,aryl groups, and heteroforms of such groups that comprise an acyl orheteroacyl group can be substituted with the substituents describedherein as generally suitable substituents for each of the correspondingcomponent of the acyl or heteroacyl group.

A carbon source sometimes comprises one or more aromatic moieties and/orheteroaromatic moieties. “Aromatic” moiety or “aryl” moiety refers to amonocyclic or fused bicyclic moiety having the well-knowncharacteristics of aromaticity; examples include phenyl and naphthyl.Similarly, “heteroaromatic” and “heteroaryl” refer to such monocyclic orfused bicyclic ring systems which contain as ring members one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits aromaticity in 5 membered rings as well as 6 membered rings.Typical heteroaromatic systems include monocyclic C5-C6 aromatic groupssuch as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl,pyrazolyl, thiazolyl, oxazolyl, and imidazolyl and the fused bicyclicmoieties formed by fusing one of these monocyclic groups with a phenylring or with any of the heteroaromatic monocyclic groups to form aC8-C10 bicyclic group such as indolyl, benzimidazolyl, indazolyl,benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl,pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like.Any monocyclic or fused ring bicyclic system which has thecharacteristics of aromaticity in terms of electron distributionthroughout the ring system is included in this definition. It alsoincludes bicyclic groups where at least the ring which is directlyattached to the remainder of the molecule has the characteristics ofaromaticity. Typically, the ring systems contain 5-12 ring member atoms.The monocyclic heteroaryls sometimes contain 5-6 ring members, and thebicyclic heteroaryls sometimes contain 8-10 ring members.

Aryl and heteroaryl moieties may be substituted with a variety ofsubstituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12aryl, C1-C8 acyl, and heteroforms of these, each of which can itself befurther substituted; other substituents for aryl and heteroaryl moietiesinclude halo, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR, NRCOR,CN, COOR, CONR₂, OOCR, COR, and NO₂, where each R is independently H,C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl,C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, C5-C10 heteroaryl,C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each R is optionallysubstituted as described above for alkyl groups. The substituent groupson an aryl or heteroaryl group may be further substituted with thegroups described herein as suitable for each type of such substituentsor for each component of the substituent. Thus, for example, anarylalkyl substituent may be substituted on the aryl portion withsubstituents typical for aryl groups, and it may be further substitutedon the alkyl portion with substituents as typical or suitable for alkylgroups.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aromatic andheteroaromatic ring systems, which are stand-alone molecules (e.g.,benzene or substituted benzene, pyridine or substituted pyridine), orwhich are bonded to an attachment point through a linking group such asan alkylene, including substituted or unsubstituted, saturated orunsaturated, cyclic or acyclic linkers. A linker often is C1-C8 alkyl ora hetero form thereof. These linkers also may include a carbonyl group,thus making them able to provide substituents as an acyl or heteroacylmoiety. An aryl or heteroaryl ring in an arylalkyl or heteroarylalkylgroup may be substituted with the same substituents described above foraryl groups. An arylalkyl group sometimes includes a phenyl ringoptionally substituted with the groups defined above for aryl groups anda C1-C4 alkylene that is unsubstituted or is substituted with one or twoC1-C4 alkyl groups or heteroalkyl groups, where the alkyl or heteroalkylgroups can optionally cyclize to form a ring such as cyclopropane,dioxolane, or oxacyclopentane. Similarly, a heteroarylalkyl group oftenincludes a C5-C6 monocyclic heteroaryl group optionally substituted withone or more of the groups described above as substituents typical onaryl groups and a C1-C4 alkylene that is unsubstituted. Aheteroarylalkyl group sometimes is substituted with one or two C1-C4alkyl groups or heteroalkyl groups, or includes an optionallysubstituted phenyl ring or C5-C6 monocyclic heteroaryl and a C1-C4heteroalkylene that is unsubstituted or is substituted with one or twoC1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groupscan optionally cyclize to form a ring such as cyclopropane, dioxolane,or oxacyclopentane.

Where an arylalkyl or heteroarylalkyl group is described as optionallysubstituted, the substituents may be on the alkyl or heteroalkyl portionor on the aryl or heteroaryl portion of the group. The substituentsoptionally present on the alkyl or heteroalkyl portion sometimes are thesame as those described above for alkyl groups, and the substituentsoptionally present on the aryl or heteroaryl portion often are the sameas those described above for aryl groups generally.

“Arylalkyl” groups as used herein are hydrocarbyl groups if they areunsubstituted, and are described by the total number of carbon atoms inthe ring and alkylene or similar linker. Thus a benzyl group is aC7-arylalkyl group, and phenylethyl is a C8-arylalkyl.

“Heteroarylalkyl” as described above refers to a moiety comprising anaryl group that is attached through a linking group, and differs from“arylalkyl” in that at least one ring atom of the aryl moiety or oneatom in the linking group is a heteroatom selected from N, O and S. Theheteroarylalkyl groups are described herein according to the totalnumber of atoms in the ring and linker combined, and they include arylgroups linked through a heteroalkyl linker; heteroaryl groups linkedthrough a hydrocarbyl linker such as an alkylene; and heteroaryl groupslinked through a heteroalkyl linker. Thus, for example,C7-heteroarylalkyl includes pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.

“Alkylene” as used herein refers to a divalent hydrocarbyl group.Because an alkylene is divalent, it can link two other groups together.An alkylene often is referred to as —(CH₂)_(n)— where n can be 1-20,1-10, 1-8, or 1-4, though where specified, an alkylene can also besubstituted by other groups, and can be of other lengths, and the openvalences need not be at opposite ends of a chain. Thus —CH(Me)- and—C(Me)₂- may also be referred to as alkylenes, as can a cyclic groupsuch as cyclopropan-1,1-diyl. Where an alkylene group is substituted,the substituents include those typically present on alkyl groups asdescribed herein.

Nitrogen may be supplied from an inorganic (e.g.,(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or glutamate).In addition to appropriate carbon and nitrogen sources, culture mediaalso can contain suitable minerals, salts, cofactors, buffers, vitamins,metal ions (e.g., Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) and othercomponents suitable for culture of microorganisms.

Engineered microorganisms sometimes are cultured in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)). In some embodiments,engineered microorganisms are cultured in a defined minimal media thatlacks a component necessary for growth and thereby forces selection of adesired expression cassette (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)). Culture media in some embodiments arecommon commercially prepared media, such as Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.). Other defined or synthetic growth mediamay also be used and the appropriate medium for growth of the particularmicroorganism are known. A variety of host organisms can be selected forthe production of engineered microorganisms. Non-limiting examplesinclude yeast (e.g., Candida tropicalis (e.g., ATCC20336, ATCC20913,ATCC20962), Yarrowia lipolytica (e.g., ATCC20228)) and filamentous fungi(e.g., Aspergillus nidulans (e.g., ATCC38164) and Aspergillusparasiticus (e.g., ATCC 24690)). In specific embodiments, yeast arecultured in YPD media (10 g/L Bacto Yeast Extract, 20 g/L Bacto Peptone,and 20 g/L Dextrose). Filamentous fungi, in particular embodiments, aregrown in CM (Complete Medium) containing 10 g/L Dextrose, 2 g/L BactoPeptone, 1 g/L Bacto Yeast Extract, 1 g/L Casamino acids, 50 mL/L 20×Nitrate Salts (120 g/L NaNO₃, 10.4 g/L KCl, 10.4 g/L MgSO₄.7H₂O), 1 mL/L1000× Trace Elements (22 g/L ZnSO₄.7H₂O, 11 g/L H₃BO₃, 5 g/L MnCl₂.7H₂O,5 g/L FeSO₄.7H₂O, 1.7 g/L CoCl₂.6H₂O, 1.6 g/L CuSO₄.5H₂O, 1.5 g/LNa₂MoO₄.2H₂O, and 50 g/L Na₄EDTA), and 1 mL/L Vitamin Solution (100 mgeach of Biotin, pyridoxine, thiamine, riboflavin, p-aminobenzoic acid,and nicotinic acid in 100 mL water).

A suitable pH range for the fermentation often is between about pH 4.0to about pH 8.0, where a pH in the range of about pH 5.5 to about pH 7.0sometimes is utilized for initial culture conditions. Depending on thehost organism, culturing may be conducted under aerobic or anaerobicconditions, where microaerobic conditions sometimes are maintained. Atwo-stage process may be utilized, where one stage promotesmicroorganism proliferation and another state promotes production oftarget molecule. In a two-stage process, the first stage may beconducted under aerobic conditions (e.g., introduction of air and/oroxygen) and the second stage may be conducted under anaerobic conditions(e.g., air or oxygen are not introduced to the culture conditions). Insome embodiments, the first stage may be conducted under anaerobicconditions and the second stage may be conducted under aerobicconditions. In certain embodiments, a two-stage process may include twomore organisms, where one organism generates an intermediate product(e.g., hexanoic acid produced by Megasphera spp.) in one stage andanother organism processes the intermediate product into a targetproduct (e.g., adipic acid) in another stage, for example.

A variety of fermentation processes may be applied for commercialbiological production of a target product. In some embodiments,commercial production of a target product from a recombinant microbialhost is conducted using a batch, fed-batch or continuous fermentationprocess, for example.

A batch fermentation process often is a closed system where the mediacomposition is fixed at the beginning of the process and not subject tofurther additions beyond those required for maintenance of pH and oxygenlevel during the process. At the beginning of the culturing process themedia is inoculated with the desired organism and growth or metabolicactivity is permitted to occur without adding additional sources (i.e.,carbon and nitrogen sources) to the medium. In batch processes themetabolite and biomass compositions of the system change constantly upto the time the culture is terminated. In a typical batch process, cellsproceed through a static lag phase to a high-growth log phase andfinally to a stationary phase, wherein the growth rate is diminished orhalted. Left untreated, cells in the stationary phase will eventuallydie.

A variation of the standard batch process is the fed-batch process,where the carbon source is continually added to the fermentor over thecourse of the fermentation process. Fed-batch processes are useful whencatabolite repression is apt to inhibit the metabolism of the cells orwhere it is desirable to have limited amounts of carbon source in themedia at any one time. Measurement of the carbon source concentration infed-batch systems may be estimated on the basis of the changes ofmeasurable factors such as pH, dissolved oxygen and the partial pressureof waste gases (e.g., CO.sub.2).

Batch and fed-batch culturing methods are known in the art. Examples ofsuch methods may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, 2.sup.nd ed., (1989) SinauerAssociates Sunderland, Mass. and Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992).

In continuous fermentation process a defined media often is continuouslyadded to a bioreactor while an equal amount of culture volume is removedsimultaneously for product recovery. Continuous cultures generallymaintain cells in the log phase of growth at a constant cell density.Continuous or semi-continuous culture methods permit the modulation ofone factor or any number of factors that affect cell growth or endproduct concentration. For example, an approach may limit the carbonsource and allow all other parameters to moderate metabolism. In somesystems, a number of factors affecting growth may be alteredcontinuously while the cell concentration, measured by media turbidity,is kept constant. Continuous systems often maintain steady state growthand thus the cell growth rate often is balanced against cell loss due tomedia being drawn off the culture. Methods of modulating nutrients andgrowth factors for continuous culture processes, as well as techniquesfor maximizing the rate of product formation, are known and a variety ofmethods are detailed by Brock, supra.

In some embodiments involving fermentation, the fermentation can becarried out using two or more microorganisms (e.g., host microorganism,engineered microorganism, isolated naturally occurring microorganism,the like and combinations thereof), where a feedstock is partially orcompletely utilized by one or more organisms in the fermentation (e.g.,mixed fermentation), and the products of cellular respiration ormetabolism of one or more organisms can be further metabolized by one ormore other organisms to produce a desired target product (e.g., adipicacid, hexanoic acid). In certain embodiments, each organism can befermented independently and the products of cellular respiration ormetabolism purified and contacted with another organism to produce adesired target product. In some embodiments, one or more organisms arepartially or completely blocked in a metabolic pathway (e.g., betaoxidation, omega oxidation, the like or combinations thereof), therebyproducing a desired product that can be used as a feedstock for one ormore other organisms. Any suitable combination of microorganisms can beutilized to carry out mixed fermentation or sequential fermentation. Anon-limiting example of an organism combination and feedstock suitablefor use in mixed fermentations or sequential fermentations where thefermented media from a first organism is used as a feedstock for asecond organism is the use of long chain dicarboxylic acids as afermentation media for Megasphaera elsdenii to produce hexanoic acid,and Candida tropicalis engineered as described herein to produce adipicacid from the hexanoic acid produced by Megasphaera elsdenii.Megasphaera elsdenii is a facultative anaerobe. Without being limited bytheory, it is believe that Megasphaera elsdenii naturally accumulateshexanoic acid as a result of anaerobic respiration. Candida tropicaliscan grow aerobically and anaerobically. In some embodiments, thehexanoic acid produced by Megasphaera elsdenii can be utilized as afeedstock for Candida tropicalis to produce adipic acid. In certainembodiments, the Megasphaera produced hexanoic acid is purified (e.g.,partially, completely) prior to being used as a feedstock for C.tropicalis.

In various embodiments adipic acid is isolated or purified from theculture media or extracted from the engineered microorganisms. In someembodiments, fermentation of feedstocks by methods described herein canproduce a target product (e.g., adipic acid) at a level of about 80% ormore of theoretical yield (e.g., 80% or more, 81% or more, 82% or more,83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% ormore, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99%or more of theoretical yield). The term “theoretical yield” as usedherein refers to the amount of product that could be made from astarting material if the reaction is 100% complete. Theoretical yield isbased on the stoichiometry of a reaction and ideal conditions in whichstarting material is completely consumed, undesired side reactions donot occur, the reverse reaction does not occur, and there are no lossesin the work-up procedure. Culture media may be tested for target product(e.g., adipic acid) concentration and drawn off when the concentrationreaches a predetermined level. Detection methods are known in the art,including but not limited to those set forth in B Stieglitz and P JWeimer, Novel microbial screen for detection of 1,4-butanediol, ethyleneglycol, and adipic acid, Appl Environ Microbiol. 198. Target product(e.g., adipic acid) may be present at a range of levels as describedherein.

A target product sometimes is retained within an engineeredmicroorganism after a culture process is completed, and in certainembodiments, the target product is secreted out of the microorganisminto the culture medium. For the latter embodiments, (i) culture mediamay be drawn from the culture system and fresh medium may besupplemented, and/or (ii) target product may be extracted from theculture media during or after the culture process is completed.Engineered microorganisms may be cultured on or in solid, semi-solid orliquid media. In some embodiments media is drained from cells adheringto a plate. In certain embodiments, a liquid-cell mixture is centrifugedat a speed sufficient to pellet the cells but not disrupt the cells andallow extraction of the media, as known in the art. The cells may thenbe resuspended in fresh media. Target product may be purified fromculture media according to known methods, such as those described inU.S. Pat. No. 6,787,669 and U.S. Pat. No. 5,296,639, for example.

In certain embodiments, target product is extracted from the culturedengineered microorganisms. The microorganism cells may be concentratedthrough centrifugation at a speed sufficient to shear the cellmembranes. In some embodiments, the cells may be physically disrupted(e.g., shear force, sonication) or chemically disrupted (e.g., contactedwith detergent or other lysing agent). The phases may be separated bycentrifugation or other method known in the art and target product maybe isolated according to known methods.

Commercial grade target product sometimes is provided in substantiallypure form (e.g., 90% pure or greater, 95% pure or greater, 99% pure orgreater or 99.5% pure or greater). In some embodiments, target productmay be modified into any one of a number of downstream products. Forexample, adipic acid may be polycondensed with hexamethylenediamine toproduce nylon. Nylon may be further processed into fibers forapplications in carpeting, automobile tire cord and clothing. Adipicacid is also used for manufacturing plasticizers, lubricant componentsand polyester polyols for polyurethane systems. Food grade adipic acidis used as a gelling aid, acidulent, leavening and buffering agent.Adipic acid has two carboxylic acid (—COOH) groups, which can yield twokinds of salts. Its derivatives, acyl halides, anhydrides, esters,amides and nitriles, are used in making downstream products such asflavoring agents, internal plasticizers, pesticides, dyes, textiletreatment agents, fungicides, and pharmaceuticals through furtherreactions of substitution, catalytic reduction, metal hydride reduction,diborane reduction, keto formation with organometallic reagents,electrophile bonding at oxygen, and condensation.

Target product may be provided within cultured microbes containingtarget product, and cultured microbes may be supplied fresh or frozen ina liquid media or dried. Fresh or frozen microbes may be contained inappropriate moisture-proof containers that may also be temperaturecontrolled as necessary. Target product sometimes is provided in culturemedium that is substantially cell-free. In some embodiments targetproduct or modified target product purified from microbes is provided,and target product sometimes is provided in substantially pure form. Incertain embodiments crystallized or powdered target product is provided.Crystalline adipic acid is a white powder with a melting point of 360°F. and may be transported in a variety of containers including one toncartons, drums, 50 pound bags and the like.

In certain embodiments, a target product (e.g., adipic acid,6-hydroxyhexanoic acid) is produced with a yield of about 0.30 grams oftarget product, or greater, per gram of glucose added during afermentation process (e.g., about 0.31 grams of target product per gramof glucose added, or greater; about 0.32 grams of target product pergram of glucose added, or greater; about 0.33 grams of target productper gram of glucose added, or greater; about 0.34 grams of targetproduct per gram of glucose added, or greater; about 0.35 grams oftarget product per gram of glucose added, or greater; about 0.36 gramsof target product per gram of glucose added, or greater; about 0.37grams of target product per gram of glucose added, or greater; about0.38 grams of target product per gram of glucose added, or greater;about 0.39 grams of target product per gram of glucose added, orgreater; about 0.40 grams of target product per gram of glucose added,or greater; about 0.41 grams of target product per gram of glucoseadded, or greater; 0.42 grams of target product per gram of glucoseadded, or greater; 0.43 grams of target product per gram of glucoseadded, or greater; 0.44 grams of target product per gram of glucoseadded, or greater; 0.45 grams of target product per gram of glucoseadded, or greater; 0.46 grams of target product per gram of glucoseadded, or greater; 0.47 grams of target product per gram of glucoseadded, or greater; 0.48 grams of target product per gram of glucoseadded, or greater; 0.49 grams of target product per gram of glucoseadded, or greater; 0.50 grams of target product per gram of glucoseadded, or greater; 0.51 grams of target product per gram of glucoseadded, or greater; 0.52 grams of target product per gram of glucoseadded, or greater; 0.53 grams of target product per gram of glucoseadded, or greater; 0.54 grams of target product per gram of glucoseadded, or greater; 0.55 grams of target product per gram of glucoseadded, or greater; 0.56 grams of target product per gram of glucoseadded, or greater; 0.57 grams of target product per gram of glucoseadded, or greater; 0.58 grams of target product per gram of glucoseadded, or greater; 0.59 grams of target product per gram of glucoseadded, or greater; 0.60 grams of target product per gram of glucoseadded, or greater; 0.61 grams of target product per gram of glucoseadded, or greater; 0.62 grams of target product per gram of glucoseadded, or greater; 0.63 grams of target product per gram of glucoseadded, or greater; 0.64 grams of target product per gram of glucoseadded, or greater; 0.65 grams of target product per gram of glucoseadded, or greater; 0.66 grams of target product per gram of glucoseadded, or greater; 0.67 grams of target product per gram of glucoseadded, or greater; 0.68 grams of target product per gram of glucoseadded, or greater; 0.69 or 0.70 grams of target product per gram ofglucose added or greater).

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology. Certain examples set forth below utilize standardrecombinant DNA and other biotechnology protocols known in the art. Manysuch techniques are described in detail in Maniatis, T., E. F. Fritschand J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. DNA mutagenesis canbe accomplished using the Stratagene (San Diego, Calif.) “QuickChange”kit according to the manufacturer's instructions.

Example 1 Cloning Hexanoate Synthase (“HexS”) Subunit Genes

Total RNA from Aspergillus parasiticus was prepared using the RiboPure™(Ambion, Austin, Tex.) kit for yeast. The genes encoding the twosubunits of hexanoate synthase (referred to as “hexA” and “hex B” wereisolated from this total RNA using the 2-step RT-PCR method withSuperscript III reverse transcriptase (Life Technologies, Carlsbad,Calif.) and the fragments were gel purified. The primers used for eachRT-PCR reaction are as follows:

HexA Aspergillus parasiticus primers SP_HexA_Apar_1_1149ATGGTCATCCAAGGGAAGAGATTGGCCGCCTCCTCTATTCAGC ASP_HexA_Apar_1_1149GTAGGCGTCACAGGAAAGACTGCGTACCA SP_HexA_Apar_941_2270TATCACCAATGCTGGATGTAAAGAAGTCGCG ASP_HexA_Apar_941_2270AATTGGGCTAGGAAACCGGGGATGC SP_HexA_Apar_2067_3016CGGTCTAATGACGGCGCATGATATCATAGCCGAAACGGTCGAG ASP_HexA_Apar_2067_3016ACTTGGCTGGAGTCCATCCCTTCGGCA SP_HexA_Apar_2812_4181CTGCCCGAGTTTGAAGTATCTCAACTTACCGCCGACGCCATG ASP_HexA_Apar_2812_4181TGAGACGCGCTGCGCAGGGC SP_HexA_Apar_3975_5016 CGAGGTGATCGAGACGCAGATGCASP_HexA_Apar_3975_5016 TTATGAAGCACCAGACATCAGCCCCAGCHexB Aspergillus parasiticus primers SP_HexB_Apar_1_1166ATGGGTTCCGTTAGTAGGGAACATGAGTCAATC ASP_HexB_Apar_1_1166GTTCCTTGTGTGAGCTCCTGAATAAGACTGCATG SP_HexB_Apar_962_2042CCATCAAAATCCCCCTCTATCACACGGGCACTGGGAGCAAC ASP_HexB_Apar_962_2042CCCACGCCTTGCGCATCTATAATCAGG SP_HexB_Apar_1837_3527TGTCCGAATATTCTCCTCGTTGTAGGTAGTGGATT ASP_HexB_Apar_1837_3527GCAGTAGTCGATAGGTACACATCCTTGGGGGTTCCATGACTGC SP_HexB_Apar_3322_4460AGAGGATCAAGGCATTATACATGAGTCTGTGGAACTTGGGCTTTCC ASP_HexB_Apar_3322_4460TTCCCCGTCCTCCATGGCCTTATGC SP_HexB_Apar_4256_5667GGCCTTTGCGCGATACGCTGGTCTCTCGGGTCCCAT ASP_HexB_Apar_4256_5667TCACGCCATTTGTTGAAGCAGGGAATG

Each of the fragments was inserted separately into the plasmid pCRBluntII (Life Technologies, Carlsbad, Calif.) such that there were four hexAplasmids, each with a different hexA gene fragment, and five hex Bplasmids each with a different hexB gene fragment. Each hexA and hexBfragment was sequence verified, after which the fragments were PCRcloned from each plasmid. Overlap PCR was then used to create the fulllength hexA and hexB genes. The hexA gene was inserted into the vectorp425GPD which has a LEU2 selectable marker and a glyceraldehyde3-phosphate dehydrogenase promoter (American Type Culture Collection)and the hexB full length gene was inserted into p426GPD which has a URA3selectable marker and a glyceraldehyde 3-phosphate dehydrogenasepromoter (American Type Culture Collection).

Example 2 Transformation of Saccharomyces cerevisiae with HexA and HexBGenes

Saccharomyces cerevisiae cells (strain BY4742, ATCC Accession Number201389) were grown in standard YPD (10 g Yeast Extract, 20 gBacto-Peptone, 20 g Glucose, 1 L total) media at about 30 degreesCelsius for about 3 days. The plasmids containing the hexA and hexBgenes were co-transformed into the Saccharomyces cerevisiae.Transformation was accomplished using the Zymo kit (Catalog numberT2001; Zymo Research Corp., Orange, Calif. 92867) using 1 μg plasmid DNAand cultured on SC drop out media with glucose (minus uracil and minusleucine) (20 g glucose; 2.21 g SC (-URA,-LEU) dry mix, 6.7 g YeastNitrogen Base, 1 L total) for 2-3 days at about 30° C.

SC(-URA) mix contains:

0.4 g Adenine hemisulfate 3.5 g Arginine   1 g Glutamic Acid 0.433 g Histidine 0.4 g Myo-Inositol 5.2 g Isoleucine 0.9 g Lysine 1.5 gMethionine 0.8 g Phenylalanine 1.1 9 Serine 1.2 g Threonine 0.8 gTryptophan 0.2 g Tyrosine 1.2 g Valine

When needed:

0.263 g Leucine  0.2 g Uracil

Co-transformants were selected and established as liquid cultures in YPDmedia under standard conditions.

Example 3 Production of Synthetic HexA and HexB Genes

Synthetic hexanoate synthase subunit genes were designed for use inCandida tropicalis. This organism uses an alternate genetic code inwhich the codon “CTG” encodes serine instead of leucine. Therefore, all“CTG” codons were replaced with the codon “TTG” to ensure that thesegenes, when translated by C. tropicalis, would generate polypeptideswith amino acid sequences identical to the wild type polypeptides foundin A. parasiticus. Due to the large size of each subunit, each wassynthesized as four fragments, and each fragment was inserted into thevector pUC57. PCR was used to clone each fragment, and overlap extensionPCR was then used to generate each full length gene.

The sequence of the synthetic gene for each hexanoate synthase subunitis set forth below. The synthetic gene encoding the hexA subunit isreferred to as hexA-AGC (“Alternate Genetic Code”) and the syntheticgene encoding the hexB subunit is referred to as hexB-AGC.

>hexA-AGC for Candida tropicalisatggtcatccaagggaagagattggccgcctcctctattcagcttctcgcaagctcgttagacgcgaagaagctttgttatgagtatgacgagaggcaagccccaggtgtaacccaaatcaccgaggaggcgcctacagagcaaccgcctctctctacccctccctcgctaccccaaacgcccaatatttcgcctataagtgcttcaaagatcgtgatcgacgatgtggcgctatctcgagtgcaaattgttcaggctcttgttgccagaaagttgaagacggcaattgctcagcttcctacatcaaagtcaatcaaagagttgtcgggtggtcggtcttctttgcagaacgagctcgtgggggatatacacaacgagttcagctccatcccggatgcaccagagcagatcttgttgcgggactttggcgacgccaacccaacagtgcaattggggaaaacgtcctccgcggcagttgccaaactaatctcgtccaagatgcctagtgacttcaacgccaacgctattcgagcccacctagcaaacaagtggggtctaggacccttgcgacaaacagcggtgttgctctacgccattgcgtcagaacccccatcgcgtttagcttcatcgagcgcagcggaagagtactgggacaacgtgtcatccatgtacgccgaatcgtgtggcatcaccctccgcccgagacaagacactatgaatgaagatgctatggcatcgtcggcgattgatccggctgtggtagccgagttttccaaggggcaccgtaggctcggagttcaacagttccaagcgctagcagaatacttacaaattgatttgtcggggtctcaagcctctcagtcggatgctttggtggcggaacttcagcagaaagtcgatctctggacggccgaaatgacccccgagtttctcgccgggatatcaccaatgttggatgtaaagaagtcgcgacgctatggctcgtggtggaacatggcacggcaggatgtcttggccttctatcgccgtccttcctacagtgaattcgtggacgacgccttggccttcaaagtttttctcaatcgtctctgtaaccgagctgatgaggccctcctcaacatggtacgcagtctttcctgtgacgcctacttcaagcaaggttctttgcccggatatcatgccgcctcgcgactccttgagcaggccatcacatccacagtggcggattgcccgaaggcacgcctcattctcccggcggtgggcccccacaccaccattacaaaggacggcacgattgaatacgcggaggcaccgcgccagggagtgagtggtcccactgcgtacatccagtctctccgccaaggcgcatctttcattggtctcaagtcagccgacgtcgatactcagagcaacttgaccgacgctttgcttgacgccatgtgcttagcactccataatggaatctcgtttgttggtaaaacctttttggtgacgggagcgggtcaggggtcaataggagcgggagtggtgcgtctattgttagagggaggagcccgagtattggtgacgacgagcagggagccggcgacgacatccagatacttccagcagatgtacgataatcacggtgcgaagttctccgagttgcgggtagttccttgcaatctagccagcgcccaagattgcgaagggttgatccggcacgtctacgatccccgtgggctaaattgggatttggatgccatccttcccttcgctgccgcgtccgactacagcaccgagatgcatgacattcggggacagagcgagttgggccaccggctaatgttggtcaatgtcttccgcgtgttggggcatatcgtccactgtaaacgagatgccggggttgactgccatccgacgcaggtgttgttgccattgtcgccaaatcacggcatcttcggtggcgatgggatgtatccggagtcaaagctagcccttgagagcttgttccatcgcatccgatcagagtcttggtcagaccagttatctatatgcggcgttcgtatcggttggacccggtcgaccggtctaatgacggcgcatgatatcatagccgaaacggtcgaggaacacggaatacgcacattttccgtggccgagatggcactcaacatagccatgttgttaacccccgactttgtggcccattgtgaagatggacctttggatgccgatttcaccggcagcttgggaacattgggtagcatccccggtttcctagcccaattgcaccagaaagtccagttggcagccgaggtgatccgtgccgtgcaggccgaggatgagcatgagagattcttgtctccgggaacaaaacctaccttgcaagcacccgtggccccaatgcacccccgcagtagccttcgtgtaggctatccccgtctccccgattatgagcaagagattcgcccgttgtccccacggttggaaaggttgcaagatccggccaatgctgtggtggtggtcgggtactcggagttggggccatggggtagcgcgcgattacggtgggaaatagagagccagggccagtggacttcagccggttatgtcgaacttgcctggttgatgaacctcatccgccacgtcaacgatgaatcctacgtcggctgggtggatactcagaccggaaagccagtgcgggatggcgagatccaggcattgtacggggaccacattgacaaccacaccggtatccgtcctatccagtccacctcgtacaacccagagcgcatggaggtcttgcaggaggtcgctgtcgaggaggatttgcccgagtttgaagtatctcaacttaccgccgacgccatgcgtctccgccatggagctaacgtttccatccgccccagtggaaatcccgacgcatgccacgtgaagcttaaacgaggcgctgttatccttgttcccaagacagttccctttgtttggggatcgtgtgccggtgagttgccgaagggatggactccagccaagtacggcatccctgagaacctaattcatcaggtcgaccccgtcacgctctatacaatttgctgcgtggcggaggcattttacagtgccggtataactcaccctcttgaggtctttcgacacattcacctctcggaactaggcaactttatcggatcctccatgggtgggccgacgaagactcgtcagctctaccgagatgtctacttcgaccatgagattccgtcggatgttttgcaagacacttatctcaacacacctgctgcctgggttaatatgctactccttggctgcacggggccgatcaaaactcccgtcggcgcatgtgccaccggggtcgagtcgatcgattccggctacgagtcaatcatggcgggcaagacaaagatgtgtcttgtgggtggctacgacgatttgcaggaggaggcatcgtatggattcgcacaacttaaggccacggtcaacgttgaagaggagatcgcctgcggtcgacagccctcggagatgtcgcgccccatggctgagagtcgtgctggctttgtcgaggcgcatggctgcggtgtacagttgttgtgtcgaggtgacatcgccttgcaaatgggtcttcctatctatgcggtcattgccagctcagccatggccgccgacaagatcggttcctcggtgccagcaccgggccagggcattctaagcttctcccgtgagcgcgctcgatccagtatgatatccgtcacgtcgcgcccgagtagccgtagcagcacatcatctgaagtctcggacaaatcatccttgacctcaatcacctcaatcagcaatcccgctcctcgtgcacaacgcgcccgatccaccactgatatggctccgttgcgagcagcgcttgcgacttgggggttgactatcgacgacttggatgtggcctcattgcacggcacctcgacgcgcggtaacgatctcaatgagcccgaggtgatcgagacgcagatgcgccatttaggtcgcactcctggccgccccttgtgggccatctgccaaaagtcagtgacgggacaccctaaagccccagcggccgcatggatgctcaatggatgcttgcaagtattggactcggggttggtgccgggcaaccgcaatcttgacacgttggacgaggccttgcgcagcgcgtctcatctctgcttccctacgcgcaccgtgcagctacgtgaggtcaaggcattcttgttgacctcatttggcttcggacagaaggggggccaagtcgtcggcgttgcccccaagtacttctttgctacgctcccccgccccgaggttgagggctactatcgcaaggtgagggttcgaaccgaggcgggtgatcgcgcctacgccgcggcggtcatgtcgcaggcggtggtgaagatccagacgcaaaacccgtacgacgagccggatgccccccgcatttttctcgatcccttggcacgtatctcccaggatccgtcgacgggccagtatcggtttcgttccgatgccactcccgccctcgatgatgatgctttgccacctcccggcgaacccaccgagctagtgaagggcatctcctccgcctggatcgaggagaaggtgcgaccgcatatgtctcccggcggcacggtgggcgtggacttggttcctctcgcctccttcgacgcatacaagaatgccatctttgttgagcgcaattatacggtaagggagcgcgattgggctgaaaagagtgcggatgtgcgcgcggcctatgccagtcggtggtgtgcaaaagaggcggtgttcaaatgtctccagacacattcacagggcgcgggggcagccatgaaagagattgagatcgagcatggaggtaacggcgcaccgaaagtcaagctccggggtgctgcgcaaacagcggcgcggcaacgaggattggaaggagtgcaattgagcatcagctatggcgacgatgcggtgatagcggtggcgttggggttgatgtctggtgcttcataa >hexB-AGC for Candida tropicalisatgggttccgttagtagggaacatgagtcaatccccatccaggccgcccagagaggcgctgcccggatctgcgctgcttttggaggtcaagggtctaacaatttggacgtgttaaaaggtctattggagttatacaagcggtatggcccagatttggatgagctactagacgtggcatccaacacgctttcgcagttggcatcttcccctgctgcaatagacgtccacgaaccctggggtttcgacctccgacaatggttgaccacaccggaggttgctcctagcaaagaaattcttgccttgccaccacgaagctttcccttaaatacgttacttagcttggcgctctattgtgcaacttgtcgagagcttgaacttgatcctgggcaatttcgatccctccttcatagttccacggggcattcccaaggcatattggcggcggtggccatcacccaagccgagagctggccaaccttttatgacgcctgcaggacggtgctccagatctctttctggattggactcgaggcttacctcttcactccatcctccgccgcctcggatgccatgatccaagattgcatcgaacatggcgagggccttctttcctcaatgctaagtgtctccgggctctcccgctcccaagttgagcgagtaattgagcacgtcaataaagggctcggagaatgcaaccgatgggttcacttggccttggttaactcccacgaaaagttcgtcttagcgggaccacctcaatccttatgggccgtttgtcttcatgtccgacggatcagagcagacaatgacctcgaccagtcgcgtatcttgttccgcaaccgaaagcctatagtggatatattatttcttcccatatccgcaccatttcacacaccgtacttggacggtgttcaagatcgcgttatcgaggctttgagctctgcttcgttggctctccattccatcaaaatccccctctatcacacgggcactgggagcaacctacaagaactacaaccacatcagctaatcccgactcttatccgcgccattaccgtggaccaattggactggccgttggtttgccggggcttgaacgcaacgcacgtgttggactttggacctggacaaacatgcagtcttattcaggagctcacacaaggaacaggtgtatcagtgatccagttgactactcaatcgggaccaaaacccgttggaggccatttggcggcagtgaactgggaggccgagtttggcttacgacttcatgccaatgtccacggtgcagctaaattgcacaaccgtatgacaacattgcttgggaagcctcctgtgatggtagccggaatgacacctactacggtgcgctgggactttgtcgctgccgttgctcaagctggataccacgtcgaattggctggtggtggctaccacgcagagcgccagttcgaggccgagattcggcgcttggcaactgccatcccagcagatcatggcatcacctgcaatctcctctacgccaagcctacgactttttcctggcagatctctgtcatcaaggatttggtgcgccagggagttcccgtggaaggaatcaccatcggcgccggcatcccttctccggaggtcgtccaagaatgtgtacagtccatcggactcaagcacatctcattcaagcctgggtctttcgaagccattcaccaagtcatacagatcgcgcgtacccatcctaactttttgatcgggttgcaatggaccgcaggacgagggggaggacatcattcctgggaagacttccatggacctattttggcaacctacgctcaaatccgatcatgtccgaatattctcctcgttgtaggtagtggattcggtggaggcccggacacgtttccctacctcacgggccaatgggcccaggcctttggctatccatgcatgcccttcgacggagtgttgctcggcagtcgcatgatggtggctcgggaagcccatacgtcagcccaggcaaaacgcttgattatagatgcgcaaggcgtgggagatgcagattggcacaagtctttcgatgagcctaccggcggcgtagtgacggtcaactcggaattcggtcaacctatccacgttctagctactcgcggagtgatgttgtggaaagaactcgacaaccgggtcttttcaatcaaagacacttctaagcgcttagaatatttgcgcaaccaccggcaagaaattgtgagccgtcttaacgcagactttgcccgtccctggtttgccgttgacggacacggacagaatgtggagttggaggacatgacctacctcgaggttctccgccgtttgtgcgatctcacgtatgtttcccaccagaagcgatgggtagatccatcatatcgaatattattgttggacttcgttcatttgcttcgagaacgattccaatgcgctattgacaaccccggcgaatatccactcgacatcatcgtccgggtggaagagagcttgaaggataaagcataccgcacgctttatccagaagatgtctctcttctaatgcatttgttcagccgacgtgacatcaagcccgtaccattcatccccaggttggatgagcgttttgagacctggtttaaaaaagactcattgtggcaatccgaagatgtggaggcggtaattggacaggacgtccagcgaatcttcatcattcaagggcctatggccgttcagtactcaatatccgacgatgagtctgttaaagacattttacacaatatttgtaatcattacgtggaggctctacaggctgattcaagagaaacttctatcggcgatgtacactcgatcacgcaaaaacctctcagcgcgtttcctgggctcaaagtgacgacaaatagggtccaagggctctataagttcgagaaagtaggagcagtccccgaaatggacgttctttttgagcatattgtcggattgtcgaagtcatgggctcggacatgtttgatgagtaaatcggtctttagggacggttctcgtttgcataaccccattcgcgccgcactccagctccagcgcggcgacaccatcgaggtgcttttaacagcagactcggaaattcgcaagattcgacttatttcacccacgggggatggtggatccacttctaaggtcgtattagagatagtctctaacgacggacaaagagttttcgccaccttggcccctaacatcccactcagccccgagcccagcgtcgtcttttgcttcaaggtcgaccagaagccgaatgagtggacccttgaggaggatgcgtctggccgggcagagaggatcaaggcattatacatgagtttgtggaacttgggctttccgaacaaggcctctgttttgggtcttaattcgcaattcacgggagaagaattgatgatcacaacggacaagattcgtgatttcgaaagggtattgcggcaaaccagtcctcttcagttgcagtcatggaacccccaaggatgtgtacctatcgactactgcgtggtcatcgcctggtctgctcttaccaagcctttgatggtctcctctttgaaatgcgacctcttggatttgctccacagcgctataagcttccactatgctccatctgtcaaaccattgcgggtgggcgatattgtcaaaacctcatcccgtatcctagcggtctcggtgagacctaggggaactatgttgacggtgtcggcggacattcagcgccagggacaacatgtagtcactgtcaaatcagatttctttctcggaggccccgttttggcatgtgaaacccctttcgaactcactgaggagcctgaaatggttgtccatgtcgactctgaagtgcgccgtgctattttacacagccgcaagtggctcatgcgagaagatcgcgcgctagatttgctagggaggcagctcctcttcagattaaagagcgaaaaattgttcaggccagacggccagctagcattgttacaggtaacaggttccgtgttcagctacagccccgatgggtcaacgacagcattcggtcgcgtatacttcgaaagcgagtcttgtacagggaacgtggtgatggacttcttgcaccgctacggtgcacctcgggcgcagttgttggagttgcaacatcccgggtggacgggcacctctactgtggcagtaagaggtcctcgacgcagccaatcctacgcacgcgtctccctcgatcataatcccatccatgtttgtccggcctttgcgcgatacgctggtctctcgggtcccattgtccatgggatggaaacctctgccatgatgcgcagaattgccgaatgggccatcggagatgcagaccggtctcggttccggagctggcatatcaccttgcaagcacccgtccaccccaacgaccctttgcgggtggagttgcagcataaggccatggaggacggggaaatggttttgaaagtacaagcatttaacgaaaggacggaagaacgcgtagcggaggcagatgcccatgttgagcaggaaactacggcttacgtcttctgtggccagggcagtcaacgacaggggatgggaatggacttgtacgtcaactgtccggaggctaaagcgttgtgggctcgcgccgacaagcatttgtgggagaaatatgggttctccatcttgcacattgtgcaaaacaaccctccagccctcactgttcactttggcagccagcgagggcgccgtattcgtgccaactatttgcgcatgatgggacagccaccgatagatggtagacatccgcccatattgaagggattgacgcggaattcgacctcgtacaccttctcctattcccaggggttgttgatgtccacccagttcgcccagcccgcattggcgttgatggaaatggctcagttcgaatggctcaaagcccagggagtcgttcagaagggtgcgcggttcgcgggacattcgttgggagaatatgccgcccttggagcttgtgcttccttcctctcatttgaagatctcatatctctcatcttttatcggggcttgaagatgcagaatgcgttgccgcgcgatgccaacggccacaccgactatggaatgttggctgccgatccatcgcggataggaaaaggtttcgaggaagcgagtttgaaatgtcttgtccatatcattcaacaggagaccggctggttcgtggaagtcgtcaactacaacatcaactcgcagcaatacgtctgtgcaggccatttccgagccctttggatgttgggtaagatatgcgatgacctttcatgccaccctcaaccggagactgttgaaggccaagagctacgggccatggtctggaagcatgtcccgacggtggagcaggtgccccgcgaggatcgcatggaacgaggtcgagcgaccattccgttgccggggatcgatatcccataccattcgaccatgttacgaggggagattgagccttatcgtgaatatttgtctgaacgtatcaaggtgggggatgtgaagccgtgcgaattggtgggacgctggatccctaatgttgttggccagcctttctccgtcgataagtcttacgttcagttggtgcacggcatcacaggtagtcctcggcttcattccttgcttcaacaaat ggcgtga

Example 4 Transformation of C. tropicalis with the Synthetic HexanoateSynthase Subunit Genes

Candida tropicalis cells (ATCC number 20962) and cultured under standardconditions in YPD medium at 30 degrees Celsius. The synthetic genesencoding hexA and hexB are amplified using standard PCR amplificationtechniques. A linear DNA construct comprising, from 5′ to 3′, the TEF(transcription elongation factor) promoter, the hexA-AGC gene, the TEFpromoter, the hexB-AGC gene, and the URA3 marker. Each end of thisconstruct is designed to contain a mini-URA-Blaster for integration ofthe construct into C. tropicalis genomic DNA (Alani E, Cao L, KlecknerN. A method for gene disruption that allows repeated use of URA3selection in the construction of multiply disrupted yeast strains.Genetics. 1987 August; 116 (4):541-545).

The construct is amplified using standard techniques. Transformation ofC. tropicalis cells with this linear construct is accomplished usingstandard electroporation techniques such as those set forth in U.S. Pat.No. 5,648,247 or 5,204,252. Transformants are selected by plating andgrowing the transformed cells on SC-URA media as described above inwhich only transformants will survive. To remove the URA cassette, theconfirmed strain is then replated onto SC complete media containing5-Fluoroorotic Acid (5-FOA) and confirmed for the loss of the URAcassette.

Example 5 Assay of Cytochrome P450 with Activity on Six Carbon Chains inC. tropicalis

Cultures of C. tropicalis are cultured in YPD media to late log phaseand then exposed to hexane exposed to various concentrations of hexaneup to about 0.1 percent (v/v) induce the expression of the cytochromep450 gene having activity specific for six carbon substrates. Afterabout 2 hours exposure to the hexane solution, cells were harvested andRNA isolated using techniques described above. The specifically inducedgene may be detected by Northern blotting and/or quantitative RT-PCR.

Cells to be analyzed for cytochrome P450 activity are grown understandard conditions and harvested for the production of microsomes.Microsomes were prepared by lysing cells in Tris-buffered sucrose (10 mMTris-HCl pH 7.5, 1 mM EDTA, 0.25M sucrose). Differential centrifugationis performed first at 25,000×g then at 100,000×g to pellet cell debristhen microsomes, respectively. The microsome pellet is resuspended in0.1M phosphate buffer (pH 7.5), 1 mM EDTA to a final concentration ofapproximately 10 mg protein/mL.

A reaction mixture containing approximately 0.3 mg microsomes, 0.1 mMsodium hexanoate, 0.7 mM NADPH, 50 mM Tris-HCl pH 7.5 in 1 mL isinitiated by the addition of NADPH and incubated at 37° C. for 10minutes. The reaction is terminated by addition of 0.25 mL 5M HCl and0.25 mL 2.5 ug/mL 10-hydroxydecanoic acid is added as an internalstandard (3.3 nmol). The mixture is extracted with 4.5 mL diethyl etherunder NaCl-saturated conditions. The organic phase is transferred to anew tube and evaporated to dryness. The residue is dissolved inacetonitrile containing 10 mM3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1 mL of 15mg/mL 18-crown-6 in acetonitril saturated with K₂CO₃. The solution isincubated at 40° C. for 30 minutes before addition of 0.05 mL 2% aceticacid. The fluorescently labeled omega-hydroxy fatty acids are resolvedvia HPLC with detection at 430 nm and excitation at 355 nm.

Example 6 Examples of Polynucleotide Regulators

Provided in the tables hereafter are non-limiting examples of regulatorpolynucleotides that can be utilized in embodiments herein. Suchpolynucleotides may be utilized in native form or may be modified foruse herein. Examples of regulatory polynucleotides include those thatare regulated by oxygen levels in a system (e.g., upregulated ordownregulated by relatively high oxygen levels or relatively low oxygenlevels).

Regulated Yeast Promoters - Up-regulated by oxygen Relative RelativeGene mRNA level mRNA level ORF name name (Aerobic) (Anaerobic) RatioYPL275W 4389 30 219.5 YPL276W 2368 30 118.4 YDR256C CTA1 2076 30 103.8YHR096C HXT5 1846 30 72.4 YDL218W 1189 30 59.4 YCR010C 1489 30 48.8YOR161C 599 30 29.9 YPL200W 589 30 29.5 YGR110W 1497 30 27 YNL237W YTP1505 30 25.2 YBR116C 458 30 22.9 YOR348C PUT4 451 30 22.6 YBR117C TKL2418 30 20.9 YLL052C 635 30 20 YNL195C 1578 30 19.4 YPR193C 697 30 15.7YDL222C 301 30 15 YNL335W 294 30 14.6 YPL036W PMA2 487 30 12.8 YML122C206 30 10.3 YGR067C 236 30 10.2 YPR192W 204 30 10.2 YNL014W 828 30 9.8YFL061W 256 30 9.1 YNR056C 163 30 8.1 YOR186W 153 30 7.6 YDR222W 196 306.5 YOR338W 240 30 6.3 YPR200C 113 30 5.7 YMR018W 778 30 5.2 YOR364W 12330 5.1 YNL234W 93 30 4.7 YNR064C 85 30 4.2 YGR213C RTA1 104 30 4 YCL064CCHA1 80 30 4 YOL154W 302 30 3.9 YPR150W 79 30 3.9 YPR196W MAL63 30 303.6 YDR420W HKR1 221 30 3.5 YJL216C 115 30 3.5 YNL270C ALP1 67 30 3.3YHL016C DUR3 224 30 3.2 YOL131W 230 30 3 YOR077W RTS2 210 30 3 YDR536WSTL1 55 30 2.7 YNL150W 78 30 2.6 YHR212C 149 30 2.4 YJL108C 106 30 2.4YGR069W 49 30 2.4 YDR106W 60 30 2.3 YNR034W SOL1 197 30 2.2 YEL073C 10430 2.1 YOL141W 81 30 1.8

Regulated Yeast Promoters - Down-regulated by oxygen Relative RelativeGene mRNA level mRNA level ORF name name (Aerobic) (Anaerobic) RatioYJR047C ANB1 30 4901 231.1 YMR319C FET4 30 1159 58 YPR194C 30 982 49.1YIR019C STA1 30 981 22.8 YHL042W 30 608 12 YHR210C 30 552 27.6 YHR079BSAE3 30 401 2.7 YGL162W STO1 30 371 9.6 YHL044W 30 334 16.7 YOL015W 30320 6.1 YCLX07W 30 292 4.2 YIL013C PDR11 30 266 10.6 YDR046C 30 263 13.2YBR040W FIG1 30 257 12.8 YLR040C 30 234 2.9 YOR255W 30 231 11.6 YOL014W30 229 11.4 YAR028W 30 212 7.5 YER089C 30 201 6.2 YFL012W 30 193 9.7YDR539W 30 187 3.4 YHL043W 30 179 8.9 YJR162C 30 173 6 YMR165C SMP2 30147 3.5 YER106W 30 145 7.3 YDR541C 30 140 7 YCRX07W 30 138 3.3 YHR048W30 137 6.9 YCL021W 30 136 6.8 YOL160W 30 136 6.8 YCRX08W 30 132 6.6YMR057C 30 109 5.5 YDR540C 30 83 4.2 YOR378W 30 78 3.9 YBR085W AAC3 451281 28.3 YER188W 47 746 15.8 YLL065W GIN11 50 175 3.5 YDL241W 58 64511.1 YBR238C 59 274 4.6 YCR048W ARE1 60 527 8.7 YOL165C 60 306 5.1YNR075W 60 251 4.2 YJL213W 60 250 4.2 YPL265W DIP5 61 772 12.7 YDL093WPMT5 62 353 5.7 YKR034W DAL80 63 345 5.4 YKR053C 66 1268 19.3 YJR147W 68281 4.1

Known and putative DNA binding motifs Regulator Known Consensus MotifAbf1 TCRNNNNNNACG Cbf1 RTCACRTG Gal4 CGGNNNNNNNNNNNCCG Gcn4 TGACTCA Gcr1CTTCC Hap2 CCAATNA Hap3 CCAATNA Hap4 CCAATNA Hsf1 GAANNTTCNNGAA Ino2ATGTGAAA Mata(A1) TGATGTANNT Mcm1 CCNNNWWRGG Mig1 WWWWSYGGGG Pho4 CACGTGRap1 RMACCCANNCAYY Reb1 CGGGTRR Ste12 TGAAACA Swi4 CACGAAA Swi6 CACGAAAYap1 TTACTAA Putative DNA Binding Motifs Best Motif (scored by E-Best Motif (scored by Regulator value) Hypergeometric) Abf1TYCGT--R-ARTGAYA TYCGT--R-ARTGAYA Ace2 RRRAARARAA-A-RARAAGTGTGTGTGTGTGTG Adr1 A-AG-GAGAGAG-GGCAG YTSTYSTT-TTGYTWTT Arg80T--CCW-TTTKTTTC GCATGACCATCCACG Arg81 AAAAARARAAAARMA GSGAYARMGGAMAAAAAAro80 YKYTYTTYTT----KY TRCCGAGRYW-SSSGCGS Ash1 CGTCCGGCGC CGTCCGGCGCAzf1 GAAAAAGMAAAAAAA AARWTSGARG-A--CSAA Bas1 TTTTYYTTYTTKY-TY-TCS-CCAATGK--CS Cad1 CATKYTTTTTTKYTY GCT-ACTAAT Cbf1 CACGTGACYACACGTGACYA Cha4 CA---ACACASA-A CAYAMRTGY-C Cin5 none none Crz1GG-A-A--AR-ARGGC- TSGYGRGASA Cup9 TTTKYTKTTY-YTTTKTY K-C-C---SCGCTACKGCDal81 WTTKTTTTTYTTTTT-T SR-GGCMCGGC-SSG Dal82 TTKTTTTYTTCTACYACA-CACAWGA Dig1 AAA--RAA-GARRAA-AR CCYTG-AYTTCW-CTTC Dot6GTGMAK-MGRA-G-G GTGMAK-MGRA-G-G Fhl1 -TTWACAYCCRTACAY-Y-TTWACAYCCRTACAY-Y Fkh1 TTT-CTTTKYTT-YTTTT AAW-RTAAAYARG Fkh2AAARA-RAAA-AAAR-AA GG-AAWA-GTAAACAA Fzf1 CACACACACACACACACSASTKCWCTCKTCGT Gal4 TTGCTTGAACGSATGCCA TTGCTTGAACGSATGCCA Gal4 (Gal)YCTTTTTTTTYTTYYKG CGGM---CW-Y--CCCG Gat1 none none Gat3RRSCCGMCGMGRCGCGCS RGARGTSACGCAKRTTCT Gcn4 AAA-ARAR-RAAAARRAR TGAGTCAYGcr1 GGAAGCTGAAACGYMWRR GGAAGCTGAAACGYMWRR Gcr2 GGAGAGGCATGATGGGGGAGGTGATGGAGTGCTCAG Gln3 CT-CCTTTCT GKCTRR-RGGAGA-GM Grf10GAAARRAAAAAAMRMARA -GGGSG-T-SYGT-CGA Gts1 G-GCCRS--TM AG-AWGTTTTTGWCAAMAHaa1 none none Hal9 TTTTTTYTTTTY-KTTTT KCKSGCAGGCWTTKYTCT Hap2YTTCTTTTYT-Y-C-KT- G-CCSART-GC Hap3 T-SYKCTTTTCYTTY SGCGMGGG--CC-GACCGHap4 STT-YTTTY-TTYTYYYY YCT-ATTSG-C-GS Hap5 YK-TTTWYYTCT-TTSMTT-YTTTCCK-C Hir1 AAAA-A-AARAR-AG CCACKTKSGSCCT-S Hir2WAAAAAAGAAAA-AAAAR CRSGCYWGKGC Hms1 AAA-GG-ARAM -AARAAGC-GGGCAC-C Hsf1TYTTCYAGAA--TTCY TYTTCYAGAA--TTCY Ime4 CACACACACACACACACACACACACACACACACACA Ino2 TTTYCACATGC SCKKCGCKSTSSTTYAA Ino4G--GCATGTGAAAA G--GCATGTGAAAA Ixr1 GAAAA-AAAAAAAARA-A CTTTTTTTYYTSGCCLeu3 GAAAAARAARAA-AA GCCGGTMMCGSYC-- Mac1 YTTKT--TTTTTYTYTTTA--TTTTTYTTKYGC Mal13 GCAG-GCAGG AAAC-TTTATA-ATACA Mal33 none none Mata1GCCC-C CAAT-TCT-CK Mbp1 TTTYTYKTTT-YYTTTTT G-RR-A-ACGCGT-R Mcm1TTTCC-AAW-RGGAAA TTTCC-AAW-RGGAAA Met31 YTTYYTTYTTTTYTYTTC Met4MTTTTTYTYTYTTC Mig1 TATACA-AGMKRTATATG Mot3 TMTTT-TY-CTT-TTTWK Msn1KT--TTWTTATTCC-C Msn2 ACCACC Msn4 R--AAAA-RA-AARAAAT Mss11TTTTTTTTCWCTTTKYC Ndd1 TTTY-YTKTTTY-YTTYT Nrg1 TTY--TTYTT-YTTTYYY Pdr1T-YGTGKRYGT-YG Phd1 TTYYYTTTTTYTTTTYTT Pho4 GAMAAAAAARAAAAR Put3CYCGGGAAGCSAMM-CCG Rap1 GRTGYAYGGRTGY Rcs1 KMAARAAAAARAAR Reb1 RTTACCCGSRfx1 AYGRAAAARARAAAARAA Rgm1 GGAKSCC-TTTY-GMRTA Rgt1 CCCTCC Rim101GCGCCGC Rlm1 TTTTC-KTTTYTTTTTC Rme1 ARAAGMAGAAARRAA Rox1YTTTTCTTTTY-TTTTT Rph1 ARRARAAAGG- Rtg1 YST-YK-TYTT-CTCCCM Rtg3GARA-AAAAR-RAARAAA Sfl1 CY--GGSSA-C Sfp1 CACACACACACACAYA Sip4CTTYTWTTKTTKTSA Skn7 YTTYYYTYTTTYTYYTTT Sko1 none Smp1AMAAAAARAARWARA-AA Sok2 ARAAAARRAAAAAG-RAA Stb1 RAARAAAAARCMRSRAAA Ste12TTYTKTYTY-TYYKTTTY Stp1 GAAAAMAA-AAAAA-AAA Stp2 YAA-ARAARAAAAA-AAM Sum1TY-TTTTTTYTTTTT-TK Swi4 RAARAARAAA-AA-R-AA Swi5 CACACACACACACACACA Swi6RAARRRAAAAA-AAAMAA Thi2 GCCAGACCTAC Uga3 GG-GGCT Yap1 TTYTTYTTYTTTY-YTYTYap3 none Yap5 YKSGCGCGYCKCGKCGGS Yap6 TTTTYYTTTTYYYYKTT Yap7 noneYfl044c TTCTTKTYYTTTT Yjl206c TTYTTTTYTYYTTTYTTT Zap1 TTGCTTGAACGGATGCCAZms1 MG-MCAAAAATAAAAS

Transcriptional repressors Associated Gene(s) Description(s) WHI5Repressor of G1 transcription that binds to SCB binding factor (SBF) atSCB target promoters in early G1; phosphorylation of Whi5p by the CDK,Cln3p/Cdc28p relieves repression and promoter binding by Whi5;periodically expressed in G1 TUP1 General repressor of transcription,forms complex with Cyc8p, involved in the establishment of repressivechromatin structure through interactions with histones H3 and H4,appears to enhance expression of some genes ROX1 Heme-dependentrepressor of hypoxic genes; contains an HMG domain that is responsiblefor DNA bending activity SFL1 Transcriptional repressor and activator;involved in repression of flocculation-related genes, and activation ofstress responsive genes; negatively regulated by cAMP-dependent proteinkinase A subunit Tpk2p RIM101 Transcriptional repressor involved inresponse to pH and in cell wall construction; required for alkalinepH-stimulated haploid invasive growth and sporulation; activated byproteolytic processing; similar to A. nidulans PacC RDR1 Transcriptionalrepressor involved in the control of multidrug resistance; negativelyregulates expression of the PDR5 gene; member of the Gal4p family ofzinc cluster proteins SUM1 Transcriptional repressor required formitotic repression of middle sporulation-specific genes; also acts asgeneral replication initiation factor; involved in telomere maintenance,chromatin silencing; regulated by pachytene checkpoint XBP1Transcriptional repressor that binds to promoter sequences of the cyclingenes, CYS3, and SMF2; expression is induced by stress or starvationduring mitosis, and late in meiosis; member of the Swi4p/Mbp1p family;potential Cdc28p substrate NRG2 Transcriptional repressor that mediatesglucose repression and negatively regulates filamentous growth; hassimilarity to Nrg1p NRG1 Transcriptional repressor that recruits theCyc8p-Tup1p complex to promoters; mediates glucose repression andnegatively regulates a variety of processes including filamentous growthand alkaline pH response CUP9 Homeodomain-containing transcriptionalrepressor of PTR2, which encodes a major peptide transporter; importedpeptides activate ubiquitin-dependent proteolysis, resulting indegradation of Cup9p and de-repression of PTR2 transcription YOX1Homeodomain-containing transcriptional repressor, binds to Mcm1p and toearly cell cycle boxes (ECBs) in the promoters of cell cycle- regulatedgenes expressed in M/G1 phase; expression is cell cycle- regulated;potential Cdc28p substrate RFX1 Major transcriptional repressor ofDNA-damage-regulated genes, recruits repressors Tup1p and Cyc8p to theirpromoters; involved in DNA damage and replication checkpoint pathway;similar to a family of mammalian DNA binding RFX1-4 proteins MIG3Probable transcriptional repressor involved in response to toxic agentssuch as hydroxyurea that inhibit ribonucleotide reductase;phosphorylation by Snf1p or the Mec1p pathway inactivates Mig3p,allowing induction of damage response genes RGM1 Putativetranscriptional repressor with proline-rich zinc fingers; overproductionimpairs cell growth YHP1 One of two homeobox transcriptional repressors(see also Yox1p), that bind to Mcm1p and to early cell cycle box (ECB)elements of cell cycle regulated genes, thereby restricting ECB-mediatedtranscription to the M/G1 interval HOS4 Subunit of the Set3 complex,which is a meiotic-specific repressor of sporulation specific genes thatcontains deacetylase activity; potential Cdc28p substrate CAF20Phosphoprotein of the mRNA cap-binding complex involved in translationalcontrol, repressor of cap-dependent translation initiation, competeswith eIF4G for binding to eIF4E SAP1 Putative ATPase of the AAA family,interacts with the Sin1p transcriptional repressor in the two-hybridsystem SET3 Defining member of the SET3 histone deacetylase complexwhich is a meiosis-specific repressor of sporulation genes; necessaryfor efficient transcription by RNAPII; one of two yeast proteins thatcontains both SET and PHD domains RPH1 JmjC domain-containing histonedemethylase which can specifically demethylate H3K36 tri- and dimethylmodification states; transcriptional repressor of PHR1; Rph1pphosphorylation during DNA damage is under control of the MEC1-RAD53pathway YMR181C Protein of unknown function; mRNA transcribed as part ofa bicistronic transcript with a predicted transcriptional repressorRGM1/YMR182C; mRNA is destroyed by nonsense-mediated decay (NMD);YMR181C is not an essential gene YLR345W Similar to6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzymes responsiblefor the metabolism of fructoso-2,6- bisphosphate; mRNA expression isrepressed by the Rfx1p-Tup1p- Ssn6p repressor complex; YLR345W is not anessential gene MCM1 Transcription factor involved in cell-type-specifictranscription and pheromone response; plays a central role in theformation of both repressor and activator complexes PHR1 DNA photolyaseinvolved in photoreactivation, repairs pyrimidine dimers in the presenceof visible light; induced by DNA damage; regulated by transcriptionalrepressor Rph1p HOS2 Histone deacetylase required for gene activationvia specific deacetylation of lysines in H3 and H4 histone tails;subunit of the Set3 complex, a meiotic-specific repressor of sporulationspecific genes that contains deacetylase activity RGT1Glucose-responsive transcription factor that regulates expression ofseveral glucose transporter (HXT) genes in response to glucose; binds topromoters and acts both as a transcriptional activator and repressorSRB7 Subunit of the RNA polymerase II mediator complex; associates withcore polymerase subunits to form the RNA polymerase II holoenzyme;essential for transcriptional regulation; target of the global repressorTup1p GAL11 Subunit of the RNA polymerase II mediator complex;associates with core polymerase subunits to form the RNA polymerase IIholoenzyme; affects transcription by acting as target of activators andrepressors

Transcriptional activators Associated Gene(s) Description(s) SKT5Activator of Chs3p (chitin synthase III), recruits Chs3p to the bud neckvia interaction with Bni4p; has similarity to Shc1p, which activatesChs3p during sporulation MSA1 Activator of G1-specific transcriptionfactors, MBF and SBF, that regulates both the timing of G1-specific genetranscription, and cell cycle initiation; potential Cdc28p substrateAMA1 Activator of meiotic anaphase promoting complex (APC/C); Cdc20pfamily member; required for initiation of spore wall assembly; requiredfor Clb1p degradation during meiosis STB5 Activator of multidrugresistance genes, forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6zinc finger domain that interacts with a PDRE (pleotropic drugresistance element) in vitro; binds Sin3p in a two-hybrid assay RRD2Activator of the phosphotyrosyl phosphatase activity of PP2A, peptidyl-prolyl cis/trans-isomerase; regulates G1 phase progression, theosmoresponse, microtubule dynamics; subunit of the Tap42p-Pph21p- Rrd2pcomplex BLM10 Proteasome activator subunit; found in association withcore particles, with and without the 19S regulatory particle; requiredfor resistance to bleomycin, may be involved in protecting againstoxidative damage; similar to mammalian PA200 SHC1 Sporulation-specificactivator of Chs3p (chitin synthase III), required for the synthesis ofthe chitosan layer of ascospores; has similarity to Skt5p, whichactivates Chs3p during vegetative growth; transcriptionally induced atalkaline pH NDD1 Transcriptional activator essential for nucleardivision; localized to the nucleus; essential component of the mechanismthat activates the expression of a set of late-S-phase-specific genesIMP2′ Transcriptional activator involved in maintenance of ionhomeostasis and protection against DNA damage caused by bleomycin andother oxidants, contains a C-terminal leucine-rich repeat LYS14Transcriptional activator involved in regulation of genes of the lysinebiosynthesis pathway; requires 2-aminoadipate semialdehyde as co-inducer MSN1 Transcriptional activator involved in regulation ofinvertase and glucoamylase expression, invasive growth and pseudohyphaldifferentiation, iron uptake, chromium accumulation, and response toosmotic stress; localizes to the nucleus HAA1 Transcriptional activatorinvolved in the transcription of TPO2, YRO2, and other genes putativelyencoding membrane stress proteins; involved in adaptation to weak acidstress UGA3 Transcriptional activator necessary for gamma-aminobutyrate(GABA)- dependent induction of GABA genes (such as UGA1, UGA2, UGA4);zinc-finger transcription factor of the Zn(2)-Cys(6) binuclear clusterdomain type; localized to the nucleus GCR1 Transcriptional activator ofgenes involved in glycolysis; DNA-binding protein that interacts andfunctions with the transcriptional activator Gcr2p GCR2 Transcriptionalactivator of genes involved in glycolysis; interacts and functions withthe DNA-binding protein Gcr1p GAT1 Transcriptional activator of genesinvolved in nitrogen catabolite repression; contains a GATA-1-type zincfinger DNA-binding motif; activity and localization regulated bynitrogen limitation and Ure2p GLN3 Transcriptional activator of genesregulated by nitrogen catabolite repression (NCR), localization andactivity regulated by quality of nitrogen source PUT3 Transcriptionalactivator of proline utilization genes, constitutively binds PUT1 andPUT2 promoter sequences and undergoes a conformational change to formthe active state; has a Zn(2)-Cys(6) binuclear cluster domain ARR1Transcriptional activator of the basic leucine zipper (bZIP) family,required for transcription of genes involved in resistance to arseniccompounds PDR3 Transcriptional activator of the pleiotropic drugresistance network, regulates expression of ATP-binding cassette (ABC)transporters through binding to cis-acting sites known as PDREs (PDRresponsive elements) MSN4 Transcriptional activator related to Msn2p;activated in stress conditions, which results in translocation from thecytoplasm to the nucleus; binds DNA at stress response elements ofresponsive genes, inducing gene expression MSN2 Transcriptionalactivator related to Msn4p; activated in stress conditions, whichresults in translocation from the cytoplasm to the nucleus; binds DNA atstress response elements of responsive genes, inducing gene expressionPHD1 Transcriptional activator that enhances pseudohyphal growth;regulates expression of FLO11, an adhesin required for pseudohyphalfilament formation; similar to StuA, an A. nidulans developmentalregulator; potential Cdc28p substrate FHL1 Transcriptional activatorwith similarity to DNA-binding domain of Drosophila forkhead but unableto bind DNA in vitro; required for rRNA processing; isolated as asuppressor of splicing factor prp4 VHR1 Transcriptional activator,required for the vitamin H-responsive element (VHRE) mediated inductionof VHT1 (Vitamin H transporter) and BIO5 (biotin biosynthesisintermediate transporter) in response to low biotin concentrations CDC20Cell-cycle regulated activator of anaphase-promoting complex/cyclosome(APC/C), which is required for metaphase/anaphase transition; directsubiquitination of mitotic cyclins, Pds1p, and other anaphase inhibitors;potential Cdc28p substrate CDH1 Cell-cycle regulated activator of theanaphase-promoting complex/cyclosome (APC/C), which directsubiquitination of cyclins resulting in mitotic exit; targets the APC/Cto specific substrates including Cdc20p, Ase1p, Cin8p and Fin1p AFT2Iron-regulated transcriptional activator; activates genes involved inintracellular iron use and required for iron homeostasis and resistanceto oxidative stress; similar to Aft1p MET4 Leucine-zippertranscriptional activator, responsible for the regulation of the sulfuramino acid pathway, requires different combinations of the auxiliaryfactors Cbf1p, Met28p, Met31p and Met32p CBS2 Mitochondrialtranslational activator of the COB mRNA; interacts with translatingribosomes, acts on the COB mRNA 5′-untranslated leader CBS1Mitochondrial translational activator of the COB mRNA; membrane proteinthat interacts with translating ribosomes, acts on the COB mRNA5′-untranslated leader CBP6 Mitochondrial translational activator of theCOB mRNA; phosphorylated PET111 Mitochondrial translational activatorspecific for the COX2 mRNA; located in the mitochondrial inner membranePET494 Mitochondrial translational activator specific for the COX3 mRNA,acts together with Pet54p and Pet122p; located in the mitochondrialinner membrane PET122 Mitochondrial translational activator specific forthe COX3 mRNA, acts together with Pet54p and Pet494p; located in themitochondrial inner membrane RRD1 Peptidyl-prolyl cis/trans-isomerase,activator of the phosphotyrosyl phosphatase activity of PP2A; involvedin G1 phase progression, microtubule dynamics, bud morphogenesis and DNArepair; subunit of the Tap42p-Sit4p-Rrd1p complex YPR196W Putativemaltose activator POG1 Putative transcriptional activator that promotesrecovery from pheromone induced arrest; inhibits both alpha-factorinduced G1 arrest and repression of CLN1 and CLN2 via SCB/MCB promoterelements; potential Cdc28p substrate; SBF regulated MSA2 Putativetranscriptional activator, that interacts with G1-specific transcriptionfactor, MBF and G1-specific promoters; ortholog of Msa2p, an MBF and SBFactivator that regulates G1-specific transcription and cell cycleinitiation PET309 Specific translational activator for the COX1 mRNA,also influences stability of intron-containing COX1 primary transcripts;localizes to the mitochondrial inner membrane; contains sevenpentatricopeptide repeats (PPRs) TEA1 Ty1 enhancer activator requiredfor full levels of Ty enhancer-mediated transcription; C6 zinc clusterDNA-binding protein PIP2 Autoregulatory oleate-specific transcriptionalactivator of peroxisome proliferation, contains Zn(2)-Cys(6) clusterdomain, forms heterodimer with Oaf1p, binds oleate response elements(OREs), activates beta- oxidation genes CHA4 DNA binding transcriptionalactivator, mediates serine/threonine activation of the catabolicL-serine (L-threonine) deaminase (CHA1); Zinc-finger protein withZn[2]-Cys[6] fungal-type binuclear cluster domain SFL1 Transcriptionalrepressor and activator; involved in repression of flocculation-relatedgenes, and activation of stress responsive genes; negatively regulatedby cAMP-dependent protein kinase A subunit Tpk2p RDS2 Zinc clustertranscriptional activator involved in conferring resistance toketoconazole CAT8 Zinc cluster transcriptional activator necessary forderepression of a variety of genes under non-fermentative growthconditions, active after diauxic shift, binds carbon source responsiveelements ARO80 Zinc finger transcriptional activator of the Zn2Cys6family; activates transcription of aromatic amino acid catabolic genesin the presence of aromatic amino acids SIP4 C6 zinc clustertranscriptional activator that binds to the carbon source- responsiveelement (CSRE) of gluconeogenic genes; involved in the positiveregulation of gluconeogenesis; regulated by Snf1p protein kinase;localized to the nucleus SPT10 Putative histone acetylase,sequence-specific activator of histone genes, binds specifically andhighly cooperatively to pairs of UAS elements in core histone promoters,functions at or near the TATA box MET28 Basic leucine zipper (bZIP)transcriptional activator in the Cbf1p- Met4p-Met28p complex,participates in the regulation of sulfur metabolism GCN4 Basic leucinezipper (bZIP) transcriptional activator of amino acid biosynthetic genesin response to amino acid starvation; expression is tightly regulated atboth the transcriptional and translational levels CAD1 AP-1-like basicleucine zipper (bZIP) transcriptional activator involved in stressresponses, iron metabolism, and pleiotropic drug resistance; controls aset of genes involved in stabilizing proteins; binds consensus sequenceTTACTAA INO2 Component of the heteromeric Ino2p/Ino4p basichelix-loop-helix transcription activator that bindsinositol/choline-responsive elements (ICREs), required for derepressionof phospholipid biosynthetic genes in response to inositol depletionTHI2 Zinc finger protein of the Zn(II)2Cys6 type, probabletranscriptional activator of thiamine biosynthetic genes SWI4 DNAbinding component of the SBF complex (Swi4p-Swi6p), a transcriptionalactivator that in concert with MBF (Mbp1-Swi6p) regulates lateG1-specific transcription of targets including cyclins and genesrequired for DNA synthesis and repair HAP5 Subunit of theheme-activated, glucose-repressed Hap2/3/4/5 CCAAT- binding complex, atranscriptional activator and global regulator of respiratory geneexpression; required for assembly and DNA binding activity of thecomplex HAP3 Subunit of the heme-activated, glucose-repressedHap2p/3p/4p/5p CCAAT-binding complex, a transcriptional activator andglobal regulator of respiratory gene expression; contains sequencescontributing to both complex assembly and DNA binding HAP2 Subunit ofthe heme-activated, glucose-repressed Hap2p/3p/4p/5p CCAAT-bindingcomplex, a transcriptional activator and global regulator of respiratorygene expression; contains sequences sufficient for both complex assemblyand DNA binding HAP4 Subunit of the heme-activated, glucose-repressedHap2p/3p/4p/5p CCAAT-binding complex, a transcriptional activator andglobal regulator of respiratory gene expression; provides the principalactivation function of the complex YML037C Putative protein of unknownfunction with some characteristics of a transcriptional activator; maybe a target of Dbf2p-Mob1p kinase; GFP- fusion protein co-localizes withclathrin-coated vesicles; YML037C is not an essential gene TRA1 Subunitof SAGA and NuA4 histone acetyltransferase complexes; interacts withacidic activators (e.g., Gal4p) which leads to transcription activation;similar to human TRRAP, which is a cofactor for c-Myc mediated oncogenictransformation YLL054C Putative protein of unknown function withsimilarity to Pip2p, an oleate- specific transcriptional activator ofperoxisome proliferation; YLL054C is not an essential gene RTG2 Sensorof mitochondrial dysfunction; regulates the subcellular location ofRtg1p and Rtg3p, transcriptional activators of the retrograde (RTG) andTOR pathways; Rtg2p is inhibited by the phosphorylated form of Mks1pYBR012C Dubious open reading frame, unlikely to encode a functionalprotein; expression induced by iron-regulated transcriptional activatorAft2p JEN1 Lactate transporter, required for uptake of lactate andpyruvate; phosphorylated; expression is derepressed by transcriptionalactivator Cat8p during respiratory growth, and repressed in the presenceof glucose, fructose, and mannose MRP1 Mitochondrial ribosomal proteinof the small subunit; MRP1 exhibits genetic interactions with PET122,encoding a COX3-specific translational activator, and with PET123,encoding a small subunit mitochondrial ribosomal protein MRP17Mitochondrial ribosomal protein of the small subunit; MRP17 exhibitsgenetic interactions with PET122, encoding a COX3-specific translationalactivator TPI1 Triose phosphate isomerase, abundant glycolytic enzyme;mRNA half- life is regulated by iron availability; transcription iscontrolled by activators Reb1p, Gcr1p, and Rap1p through binding sitesin the 5′ non-coding region PKH3 Protein kinase with similarity tomammalian phosphoinositide- dependent kinase 1 (PDK1) and yeast Pkh1pand Pkh2p, two redundant upstream activators of Pkc1p; identified as amulticopy suppressor of a pkh1 pkh2 double mutant YGL079W Putativeprotein of unknown function; green fluorescent protein (GFP)- fusionprotein localizes to the endosome; identified as a transcriptionalactivator in a high-throughput yeast one-hybrid assay TFB1 Subunit ofTFIIH and nucleotide excision repair factor 3 complexes, required fornucleotide excision repair, target for transcriptional activators PET123Mitochondrial ribosomal protein of the small subunit; PET123 exhibitsgenetic interactions with PET122, which encodes a COX3 mRNA- specifictranslational activator MHR1 Protein involved in homologousrecombination in mitochondria and in transcription regulation innucleus; binds to activation domains of acidic activators; required forrecombination-dependent mtDNA partitioning MCM1 Transcription factorinvolved in cell-type-specific transcription and pheromone response;plays a central role in the formation of both repressor and activatorcomplexes EGD1 Subunit beta1 of the nascent polypeptide-associatedcomplex (NAC) involved in protein targeting, associated with cytoplasmicribosomes; enhances DNA binding of the Gal4p activator; homolog of humanBTF3b STE5 Pheromone-response scaffold protein; binds Ste11p, Ste7p, andFus3p kinases, forming a MAPK cascade complex that interacts with theplasma membrane and Ste4p-Ste18p; allosteric activator of Fus3p thatfacilitates Ste7p-mediated activation RGT1 Glucose-responsivetranscription factor that regulates expression of several glucosetransporter (HXT) genes in response to glucose; binds to promoters andacts both as a transcriptional activator and repressor TYE7 Serine-richprotein that contains a basic-helix-loop-helix (bHLH) DNA binding motif;binds E-boxes of glycolytic genes and contributes to their activation;may function as a transcriptional activator in Ty1-mediated geneexpression VMA13 Subunit H of the eight-subunit V1 peripheral membranedomain of the vacuolar H+-ATPase (V-ATPase), an electrogenic proton pumpfound throughout the endomembrane system; serves as an activator or astructural stabilizer of the V-ATPase GAL11 Subunit of the RNApolymerase II mediator complex; associates with core polymerase subunitsto form the RNA polymerase II holoenzyme; affects transcription byacting as target of activators and repressors VAC14 Protein involved inregulated synthesis of PtdIns(3,5)P(2), in control of trafficking ofsome proteins to the vacuole lumen via the MVB, and in maintenance ofvacuole size and acidity; interacts with Fig4p; activator of Fab1p

Example 7 Cloning of HEXA and HEXB Genes

Aspergillus parasiticus (ATCC 24690) cultures were grown in malt extractbroth media (15 g/L malt extract broth, Difco) with shaking at 25° C.for 3 days. A. parasiticus pellets were transferred to a 1.5 mL tube toprovide a volume of pellets equal to approximately 500 uL. The myceliawere frozen in a dry ice ethanol bath, transferred to a mortar andpestle, and ground into a fine powder. The powder was placed in a 1.5 mLtube with approximately 500 uL 0.7 mm Zirconia beads, and total RNA wasprepared using a Ribopure Plant Kit (Ambion), according tomanufacturer's recommendations.

First strand synthesis of cDNA was performed with gene-specific primersoAA0031 (for HEXA) and oAA0041 (for HEXB) in a reaction containing 0.2uL of gene-specific primer (10 uM), 300 ng total RNA, 1.0 uL dNTP (10mM), and sterile water to bring the volume to 13 uL. The totalRNA/primer mixtures were heated at 65° C. for 5 minutes then cooled onice for 5 minutes before the addition of 4 uL 5× First strand buffer, 1uL 0.1M DTT, 1 uL H₂O, and 1 uL Superscript III RT (Invitrogen). Firststrand synthesis reactions were incubated at 55° C. for 1 hour, followedby inactivation of the enzyme at 70° C. for 15 minutes and cooling ofthe reactions to 4° C. The primers utilized for isolation of HEXA andHEXB genes were configured to independently amplify the HEXA and HEXBgenes in fragments, having fragment lengths in the range of betweenabout 1.0 kilobases (kb) to about 1.6 kb, with approximately 200 bp ofoverlapping sequence between the fragments. The sequences are shown inthe tables below.

Oligonucleotides for cloning of HEXA DNA fragments HEXA PCR productOligos Sequence sequence (bp) oAA0022ATGGTCATCCAAGGGAAGAGATTGGCCGCCTCCTCTATTCAGC    1-1149 1149 oAA0023GTAGGCGTCACAGGAAAGACTGCGTACCA oAA0024 TATCACCAATGCTGGATGTAAAGAAGTCGCG 941-2270 1330 oAA0025 AATTGGGCTAGGAAACCGGGGATGC o0AA0026CGGTCTAATGACGGCGCATGATATCATAGCCGAAACGGTCGAG 2067-3016 950 oAA0027ACTTGGCTGGAGTCCATCCCTTCGGCA oAA0028CTGCCCGAGTTTGAAGTATCTCAACTTACCGCCGACGCCATG 2812-4181 1370 oAA0029TGAGACGCGCTGCGCAGGGC oAA0030 CGAGGTGATCGAGACGCAGATGC 3975-5016 1042oAA0031 TTATGAAGCACCAGACATCAGCCCCAGC oAA0046gtactagtaaaaaaATGGTCATCCAAGGGAAGAGATTGGCCGCCT    1-5016 5041CCTCTATTCAGC oAA0047 gtcccgggctaTTATGAAGCACCAGACATCAGCCCCAGC oAA0051tacccgggctattagtgatggtggtgatggtgTGAAGCACCAGAC    1-5016 5062ATCAGCCCCAGC

Oligonucleotides for cloning of HEXB DNA fragments HEXB PCR productOligos Sequence sequence (bp) oAA0032 ATGGGTTCCGTTAGTAGGGAACATGAGTCAATC   1-1166 1166 oAA0033 GTTCCTTGTGTGAGCTCCTGAATAAGACTGCATG oAA0034CCATCAAAATCCCCCTCTATCACACGGGCACTGGGAGCAAC  962-2042 1081 oAA0035CCCACGCCTTGCGCATCTATAATCAGG oAA0036 TGTCCGAATATTCTCCTCGTTGTAGGTAGTGGATT1837-3527 1691 oAA0037 GCAGTAGTCGATAGGTACACATCCTTGGGGGTTCCATGACTGCoAA0038 AGAGGATCAAGGCATTATACATGAGTCTGTGGAACTTGGGCTTTCC 3323-4461 1139oAA0039 TTCCCCGTCCTCCATGGCCTTATGC oAA0040GGCCTTTGCGCGATACGCTGGTCTCTCGGGTCCCAT 4256-5667 1412 oAA0041TCACGCCATTTGTTGAAGCAGGGAATG oAA0048gtactagtaaaaaaATGGGTTCCGTTAGTAGGGAACATGAGTCAA    1-5667 5694 TC oAA0049gtgtttaaacctaTCACGCCATTTGTTGAAGCAGGGAATG oAA0111ggtttaaacctatcagtgatggtggtgatggtgCGCCATTTGTTG    1-5667 5714AAGCAGGGAATGAA

HEXA and HEXB gene fragments were PCR amplified using the cDNA generatedabove by the addition of 5 uL 10×Pfu reaction buffer, 1.0 uL dNTPs (10mM), 1.0 uL Sense and Antisense Primer Mix (10 uM), 1.0 uL Pfu UltraFusion HS (Agilent), 2.0 uL cDNA, 40 uL sterile H₂O. Thermocyclingparameters used to amplify the HEXA and HEXB genes were 94° C. for 5minutes, 40 cycles of 94° C. 30 seconds, 62° C. 40 seconds, 72° C. 4minutes, followed by 72° C. 10 minutes and a 4° C. hold. PCR products ofthe correct size were gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmcorrect DNA sequence.

DNA fragments of HEXA and HEXB were PCR amplified using thesequence-confirmed fragments in pCR-BluntII as template in order toproduce overlapping DNA fragments covering the entire sequence of bothHEXA and HEXB. The overlapping DNA fragments for each gene were combinedin a 50 uL overlap extension PCR reaction containing each DNA fragmentat 0.2 nM, sense and antisense primers at 0.2 uM each, 1×Pfu reactionbuffer, 1.0 uL Pfu Ultra Fusion HS polymerase, and 0.2 mM dNTPs. Uniquerestriction sites were incorporated into the sense and antisense primersto allow for cloning the HEXA and HEXB genes into p425GPD and p426GPDrespectively. For HEXA the restriction sites were SpeI/SmaI and for HEXBthe restriction sites were SpeI/PmeI. Ligation of the HEXA and HEXBgenes into p425GPD and p426GPD resulted in plasmids pAA020 and pAA021respectively. Variants of the HEXA and HEXB genes that incorporatedC-terminal 6×His tags were constructed by using an antisense primerencoding a 6×His sequence. Ligation of the HEXA-6×His and HEXB-6×Hisgenes into p425GPD and p426GPD resulted in plasmids pAA031 and pAA032,respectively. Vectors pAA020, pAA021, pAA031 and pAA032 were used todemonstrate protein expression in S. cerevisiae, as shown in FIGS. 11and 12.

Example 8 Cloning of STCJ and STCK Genes

Total RNA was prepared from Aspergillus nidulans (ATCC 38163), asdescribed in Example 1. First strand synthesis of cDNA was performedwith gene-specific primers oAA0008 (for STCJ) and oAA0021 (for STCK) ina reaction containing 0.2 uL of gene-specific primer (10 uM), 300 ngtotal RNA, 1.0 uL dNTP (10 mM), and sterile water to bring the volume to13 uL. The total RNA/primer mixtures were heated at 65° C. for 5 minutesthen cooled on ice for 5 minutes before the addition of 4 uL 5× Firststrand buffer, 1 uL 0.1M DTT, 1 uL H₂O, and 1 uL Superscript III RT(Invitrogen). First strand synthesis reactions were incubated at 55° C.for 1 hour, followed by inactivation of the enzyme at 70° C. for 15minutes and cooling of the reactions to 4° C. Primers design strategiessubstantially similar to those described herein were used to amplify theSTCJ and STCK genes in fragments in the range of between about 1.1 kb toabout 1.6 kb, with approximately 200 bp of overlapping sequence betweenthe fragments. The primers used to amplify the STCJ and STCK genes areshown in the tables below.

Oligonucleotides for cloning of STCJ DNA fragments STCJ PCR productOligos Sequence sequence (bp) oAA0001 ATGACCCAAAAGACTATACAGCAGGTCCCAAGA   1-1290 1290 oAA0002 TATGGTGCATCGAATGTTGTTTGCCTGG oAA0009AAAATGCGTGAGCACTTTGTCCAGCGC 1021-2506 1486 oAA0004CGACGTAATTGACGTTGTCAACATGCCG oAA0005 CATCTCGGGTTCCCATCACTCCCTGAGTATGAC2284-3424 1141 oAA0006 GACAAAGAAGCTGGACACCGCAGCCTTGGGATTCCACGAAC oAA0007GATCTGCCTTGTCGGTGGCTATGACGACCTTCAGCCTGAGGAGTCA 3234-4680 1447 oAA0008TTAACGGATGATAGAGGCCAACGGCCAAAGACACCACTTGCGTACAC oAA0126cacacaactagtaaaaaaATGACCCAAAAGACTATACAGCAGGTCC    1-4680 4710 CAAGAoAA0127 tgtgtgcccgggTTAACGGATGATAGAGGCCAACGGCCAAAGACAC CACTTGCGTACACoAA0154 tacccgggctattagtgatggtggtgatggtgACGGATGATAGAGG    1-4680 4730CCAAC

Oligonucleotides for cloning of STCK DNA fragments STCK PCR productOligos Sequence sequence (bp) oAA0012 ATGACTCCATCACCGTTTCTCGATGCTGT   1-1110 1110 oAA0013 CACATGGGTAGCATCGTTCATTGCCCAACACAAAGCGGGCCAGTTAACTC oAA0014 GTCGAGCTAAGAGTGACTGATGCCATTGGC  901-2510 1610 oAA0015CGTAATTCAGCTTCTGAACCTGAGCCCAGG oAA0016 CTTTGCCCGGCCGTGGTTCGC 2301-35551255 oAA0017 CCCCCAAGCTCGACAACGGGC oAA0018TTCTCAAAATGCACCGGACTGATTACTTGGA 3350-4682 1333 oAA0019CCCATTCCTCTCTCCTGCGTGCCCTGGCCGGTAAAGACGTAT oAA0020CCCTCCTTCGATGGACTTGTCCGGGCAAACGACCGGTTGCGAATGG 4477-5745 1268 AGAToAA0021 CTACCTATTCTCTTCAACCCGCCGTAACAGC oAA0128cacacaactagtaaaaaaATGACTCCATCACCGTTTCTCGATGCTGT    1-5745 5775 oAA0129tgtgtgcccgggCTACCTATTCTCTTCAACCCGCCGTAACAGC oAA0170tgtgtgcccgggctatcagtgatggtggtgatggtgCCTATTCTCT    1-5745 5796 TCAAC

STCJ and STCK fragments were amplified using the cDNA prepared above inPCR reactions containing 5 uL 10×Pfu reaction buffer, 1.0 uL dNTPs (10mM), 1.0 uL Sense and Antisense Primer Mix (10 uM), 1.0 uL Pfu UltraFusion HS (Agilent), 2.0 uL cDNA, 40 uL sterile H₂O. Thermocyclingparameters used were 94° C. for 5 minutes, 40 cycles of 94° C. 30seconds, 62° C. 40 seconds, 72° C. 4 minutes, followed by 72° C. 10minutes and a 4° C. hold. PCR products of the correct size were gelpurified and ligated into pCR-Blunt II-TOPO (Invitrogen), transformedinto competent TOP10 E. coli cells (Invitrogen). PCR inserts weresequenced to confirm the correct DNA sequence. DNA fragments of STCJ andSTCK were PCR amplified using the sequence-confirmed fragments inpCR-BluntII as template in order to produce overlapping DNA fragmentscovering the entire sequence of both STCJ and STCK. The overlapping DNAfragments for each gene were combined in a 50 uL overlap extension PCRreaction containing each DNA fragment at 0.2 nM, sense and antisenseprimers at 0.2 uM each, 1×Pfu reaction buffer, 1.0 uL Pfu Ultra FusionHS polymerase, and 0.2 mM dNTPs.

Sense and antisense primers were designed to incorporate uniquerestriction sites for cloning the STCJ and STCK genes into p425GPD andp426GPD respectively. For STCJ the restriction sites were SpeI/XmaI andfor STCK the restriction sites were SpeI/SmaI. Ligation of the STCJ andSTCK genes into p425GPD and p426GPD resulted in plasmids pAA040 andpAA042 respectively. Variants of the STCJ and STCK genes thatincorporated C-terminal 6×His tags were constructed by using anantisense primer encoding a 6×His sequence. Ligation of the STCJ-6×Hisand STCK-6×His genes into p425GPD and p426GPD resulted in plasmidspAA041 and pAA043. Vectors pAA040, pAA0421 pAA042 and pAA043 were usedto demonstrate protein expression in S. cerevisiae, as shown in FIGS. 11and 12.

Example 9 Design and Cloning of HEXA and HEXB Genes for C. tropicalisAlternate Genetic Code

The HEXA and HEXB genes contain multiple CTG codons, which normally codefor leucine. However, certain organisms, Candida tropicalis for example,translate CTG as serine. DNA sequences for HEXA and HEXB were preparedthat replaced all CTG codons with TTG codons, which is translated asleucine in C. tropicalis. The TTG codon was chosen due to it being themost frequently used leucine codon in C. tropicalis. The alternategenetic code (AGC) HEXA and HEXB genes were synthesized as equal sizefragments with 200 bp overlaps and ligated into pUC57 vector (IntegratedDNA Technologies). DNA fragments of AGC-HEXA and AGC-HEXB were PCRamplified using the fragments in pUC57 as template in order to produceoverlapping DNA fragments covering the entire sequence of both AGC-HEXAand AGC-HEXB.

The overlapping DNA fragments for each gene were combined in a 50 uLoverlap extension PCR reaction containing each DNA fragment at 0.2 nM,sense and antisense primers at 0.2 uM each, 1×Pfu reaction buffer, 1.0uL Pfu Ultra Fusion HS polymerase, and 0.2 mM dNTPs. Sense and antisenseprimers incorporated unique Sapl restriction sites for cloning theAGC-HEXA and AGC-HEXB genes into pAA105 resulting in plasmids pAA127 andpAA129 respectively. Gene variants of AGC-HEXA and AGC-HEXB thatcontained C-terminal 6×His tags were ligated into pAA105 resulting inplasmids pAA128 and pAA130 respectively. The alternate genetic codeprimers used to alter leucine codons for C. tropicalis expression ofHEXA and HEXB are shown in the tables below.

Oligonucleotides for cloning of AGC-HEXA DNA fragments AGC- HEXAPCR product Oligos Sequence sequence (bp) oAA0383cacacagctcttctagaATGGTCATCCAAGGGAAGAG    1-1404 1421 oAA0055AGTATCGACGTCGGCTGACTTGAGACCA oAA0056 CCATCACATCCACAGTGGCGG 1205-26091405 oAA0057 AACCAGGCAAGTTCGACATAACCGGC oAA0058GTAGGCTATCCCCGTCTCCCCGATTATG 2410-3814 1405 oAA0059TGATTGAGGTCAAGGATGATTTGTCCGAGA oAA0060 TCTTCCTATCTATGCGGTCATTGCCAGCT3615-5016 1419 oAA0384 cacacagctcttcctttTTATGAAGCACCAGACATCAAC oAA0385cacacagctcttcctttttagtgatggtggtgatggtgTGAAGCAC    1-5016 5071CAGACATCAACCCCAACG

Oligonucleotides for cloning of AGC-HEXB DNA fragments AGC- PCR HEXBproduct Oligos Sequence sequence (bp) oAA0386 cacacagctcttctagaATGGGT   1-1566 1583 TCCGTTAGTAGGGA oAA0064 CAAATCCTTGATGACAGAGA TCTGCCAGGAoAA0065 GCTGGGACTTTGTCGCTGCCGT 1367-2933 1567 TGCTCAAGCTGGAT oAA0066ACTGCTCCTACTTTCTCGAACTT ATAGAGCCCTTG oAA0067 ATATCCGACGATGAGTCTGT2734-4299 1566 oAA0068 ATGGACAATGGGACCCG AGA oAA0069GGACTTCTTGCACCGCTACG 4101-5667 1584 oAA0387 cacacagctcttcctttTCACGCCATTTGTTGAAGCAAAG oAA0388 cacacagctcttccttttcagtga    1-5667 5692tggtggtgatggtgCGCCATTTG TTGAAGCA

Example 10 Transformation of S. cerevisiae Procedure

Competent cells of S. cerevisiae strain BY4742 were prepared using theFrozen-EZ Yeast Transformation II Kit (Zymo Research) followingmanufacturer's instructions. 50 uL aliquots of competent cells werestored at −80° C. until use. Competent cells were transformed by theaddition of 0.5-1.0 ug of intact plasmid DNA as instructed by theFrozen-EZ Yeast Transformation II Kit (Zymo Research). Selection fortransformants was performed by plating on selective media; SC-URA (forp426-based vectors) or SC-LEU (for p425-based vectors).

Example 11 Transformation of C. tropicalis Procedure

5 mL YPD start cultures were inoculated with a single colony of C.tropicalis and incubated overnight at 30° C., with shaking at about 200rpm. The following day, fresh 25 mL YPD cultures, containing 0.05%Antifoam B, were inoculated to an initial OD_(600nm) of 0.4 and theculture incubated at 30° C., with shaking at about 200 rpm until anOD_(600nm) of 1.0-2.0 was reached. Cells were pelleted by centrifugationat 1,000×g, 4° C. for 10 minutes. Cells were washed by resuspending in10 mL sterile water, pelleted, resuspended in 1 mL sterile water andtransferred to a 1.5 mL microcentrifuge tube. The cells were then washedin 1 mL sterile TE/LiOAC solution, pH 7.5, pelleted, resuspended in 0.25mL TE/LiOAC solution and incubated with shaking at 30° C. for 30minutes.

The cell solution was divided into 50 uL aliquots in 1.5 mL tubes towhich was added 5-8 ug of linearized DNA and 5 uL of carrier DNA (boiledand cooled salmon sperm DNA, 10 mg/mL). 300 uL of sterile PEG solution(40% PEG 3500, 1×TE, 1× LiOAC) was added, mixed thoroughly and incubatedat 30° C. for 60 minutes with gentle mixing every 15 minutes. 40 uL ofDMSO was added, mixed thoroughly and the cell solution was incubated at42° C. for 15 minutes. Cells were then pelleted by centrifugation at1,000×g 30 seconds, resuspended in 500 uL of YPD media and incubated at30° C. with shaking at about 200 rpm for 2 hours. Cells were thenpelleted by centrifugation and resuspended in 1 mL 1×TE, cells werepelleted again, resuspended in 0.2 mL 1×TE and plated on selectivemedia. Plates were incubated at 30° C. for growth of transformants.

Example 12 HEXA and HEXB Expression in S. cerevisiae

Plasmids pAA031 and pAA032 were transformed into competent BY4742 S.cerevisiae cells independently and in combination. Selection fortransformants containing pAA031 was performed on SC-LEU plates.Selection for transformants containing pAA032 was performed on SC-URAplates. Selection for transformants containing both pAA031 and pAA032was performed on SC-URA-LEU plates. Single colonies were used toinoculate 5 mL of SC drop out media and grown overnight at 30° C., withshaking as described herein. Cells from 3 mL of overnight culture wereharvested by centrifugation at 12,000 rpm for 2 minutes. Cell pelletswere incubated at −80° C. until frozen.

Approximately 500 uL of cold 0.7 mm zirconia beads (Ambion) were addedon top of the frozen cell pellets. Yeast lysis buffer (50 mM Tris pH8.0, 0.1% Triton X100, 0.5 mM EDTA, 1× ProCEASE protease inhibitors [GBiosciences]) was added to fill the tube leaving as little air in thetube as possible, the tubes were placed on ice during manipulations.Cells were broken using three, 2 minute cycles in a Bead Beater(BioSpec) with 1 minute rests on ice between cycles. 200 uL of wholecell extract (WCE) was removed to a new tube and the remainder of thewhole cell extract was centrifuged at 16,000×g, 4° C. for 15 minutes topellet insoluble debris. The supernatant was removed to a new tube asthe soluble cell extract (SCE). The protein content in the soluble cellextract was determined by Bradford assay (Pierce). A volume of SCEcontaining 50 ug of protein (and the same volume WCE) was precipitatedby the addition of 4 volumes of cold 100% acetone. After centrifugationat 16,000×g, and 4° C. for 15 minutes, the supernatant was carefullyremoved and the pellet washed with 200 uL of cold 80% acetone andcentrifuged again. The supernatant again was carefully removed and thecell pellets air dried for 5 minutes.

Protein pellets were then resuspended in 1×LDS sample buffer containing50 mM DTT (Invitrogen) by incubating at 70° C., with shaking at about1200 rpm. After brief centrifugation and cooling to room temperature,samples (20 ug) were separated by SDS PAGE and transferred tonitrocellulose for immunodetection with mouse anti-6×His antibodies(Abcam). Incubation in 1:5,000 primary antibody was performed overnightat room temperature, incubation in 1:5,000 donkey anti-mouse HRPconjugate secondary antibody was performed for 3 hours at roomtemperature, and detection was performed with SuperSignal West Picochemiluminescent substrate (Pierce). Multiple clones displayed solubleexpression of both HEXA and HEXB subunits of hexanoate synthase. Asshown in FIG. 11, a substantial portion of the expressed proteinfractionated with the insoluble pellet. Strains sAA061, sAA140, sAA141,sAA142 contained 6×His-tagged HEXA and HEXB proteins. Strain sAA048contained only vectors p425GPD and p426GPD.

Example 13 STCJ and STCK Expression in S. cerevisiae

Plasmids pAA041 and pAA043 were cotransformed into competent BY4742 S.cerevisiae. Selection for transformants containing both pAA041 andpAA043 was performed on SC-URA-LEU plates. Culture growth, cell extractpreparation, SDS PAGE, and immunodetection were performed as describedherein. One clone displayed soluble expression of both STCJ and STCKsubunits. As shown in FIG. 11, a substantial portion of the expressedprotein fractionated with the insoluble pellet. Strain sAA144 contained6×His-tagged STCJ and STCK proteins. Strain sAA048 contained onlyvectors p425GPD and p426GPD.

Example 14 HEXA and HEXB Expression in C. tropicalis

Plasmids pAA128 and pAA130 were linearized using Clal, and cotransformedinto competent sAA103 cells (ura3/ura3, pox4::ura3/pox4::ura3,pox5::ura3/pox5::ura3). The Clal recognition sites in the HEXA and HEXBORF's are blocked due to overlapping dam methylation. Selection fortransformants containing integrated vector DNA was performed on SC-URAplates. Confirmation of vector integration was performed by PCR usingHEXA and HEXB specific primers. Transformants that were PCR positive forboth HEXA and HEXB were selected for analysis of target proteinexpression. Overnight culture growth was performed as described herein.Fresh 5 mL YPD cultures were inoculated from the overnight cultures toan initial OD_(600nm) of 0.4 and incubated until the OD_(600nm) reached˜5-8, at which point the culture was harvested. Cell extractpreparation, SDS PAGE, and immunodetection were performed as describedherein. Strains sAA269 and sAA270 contained plasmids pAA128 and pAA130integrated into the genome for expression of 6×His-tagged HEXA and HEXBproteins. Both strains displayed soluble expression of 6×His-tagged HEXAand HEXB subunits as shown in FIG. 12. 6×His tagged HEXA and HEXBexpressed in strains sAA269 and sAA270 are indicated with arrows. 6×Histagged STCJ and STCK from strain sAA144 were included as a positivecontrol. Strain sAA103 is the parent strain for sAA269 and sAA270 anddoes not contain integrated vectors for the expression of 6×His-taggedHEXA and HEXB.

Example 15 Procedure for Recycling of the URA3 Marker

C. tropicalis has a limited number of selectable marker, as compared toS. cerevisiae, therefore, the URA3 marker is “recycled” to allowmultiple rounds of selection using URA3. To reutilize the URA3 markerfor subsequent engineering of C. tropicalis, a single colony having theUra⁺ phenotype was inoculated into 3 mL YPD and grown overnight at 30°C. with shaking. The overnight culture was then harvested bycentrifugation and resuspended in 1 mL YNB+YE (6.7 g/L Yeast NitrogenBroth, 3 g/L Yeast Extract). The resuspended cells were then seriallydiluted in YNB+YE and 100 uL aliquots plated on YPD plates (incubationovernight at 30° C.) to determine titer of the original suspension.Additionally, triplicate 100 uL aliquots of the undiluted suspensionwere plated on SC Dextrose (Bacto Agar 20 g/L, Uracil 0.3 g/L, Dextrose20 g/L, Yeast Nitrogen Broth 6.7 g/L, Amino Acid Dropout Mix 2.14 g/L)and 5-FOA. at 3 different concentrations (0.5, 0.75, 1 mg/mL).

Plates were incubated for at least 5 days at 30° C. Colonies arising onthe SC Dextrose+5-FOA plates were resuspended in 50 uL sterile,distilled water and 5 uL utilized to streak on to YPD and SC-URA (SCDextrose medium without Uracil) plates. Colonies growing only on YPD andnot on SC-URA plates were then inoculated into 3 mL YPD and grownovernight at 30° C. with shaking. Overnight cultures were harvested bycentrifugation and resuspended in 1.5 mL YNB (6.7 g/L Yeast NitrogenBroth). The resuspended cells were serially diluted in YNB and 100 uLaliquots plated on YPD plates and incubation overnight at 30° C. todetermine initial titer. 1 mL of each undiluted cell suspension also wasplated on SC-URA and incubated for up to 7 days at 30° C. Colonies onthe SC-URA plates are revertants and the isolate with the lowestreversion frequency (<10⁻⁷) was used for subsequent strain engineering.

Example 16 Omega Oxidation of Hexane and Hexanoic Acid to Adipic Acid

Starter cultures of strain sAA003 were grown in YPD_(2.0) (1% yeastextract, 2% peptone, 2% dextrose) overnight as described. Startercultures were used to inoculate 100 mL of fresh YPD_(2.0) to an initialOD_(600nm) of 0.4 and incubated overnight at 30° C., with shaking atabout 200 rpm. The 100 mL culture was pelleted by centrifugation at4,000×g, 23° C. for 10 minutes and resuspended in 100 mL fresh YPD_(0.1)media (1% yeast extract, 1% peptone, 0.1% dextrose). The culture wasdivided into 4×25 mL cultures to which were added either 1% hexane,0.05% hexanoic acid, 1.0% hexanoic acid, or no other carbon source.Strain sAA003 is completely blocked in β-oxidation, thereforefermentation tested the ability of the β-oxidation pathway to oxidize C6substrates. Samples were taken at 24, 48, and 72 hours and analyzed byLC-MS (Scripps Center for Mass Spectrometry) using published methods forthe detection of adipic acid (Cheng et al., 2000). The data for the 72hour time-point, shown in the table below demonstrates that strainsAA003 was able to oxidize both hexanoic acid and hexane to adipic acid.The results also indicate that the 1% hexanoic acid level was toxic tothe cells leading to no production of adipic acid over backgroundlevels.

Oxidation of hexane and hexanoic acid to adipic acid Time (h) MEDIAAdipic acid (mg/L) 0 YPD_(0.1) 0.000 72 YPD_(0.1) 0.005 72 YPD_(0.1) +0.05% Hexanoic Acid 0.406 72 YPD_(0.1) + 1% Hexanoic Acid 0.003 72YPD_(0.1) + 1% Hexane 0.091

Example 17 Identification of P450 Alleles Induced by Exposure to Hexaneor Hexanoic Acid

350 mL cultures grown overnight in YNB-Salts+2.0% Glucose (6.7 g/L YeastNitrogen Broth, 3.0 g/L Yeast Extract, 3.0 g/L ammonium sulfate, 3.0 g/Lmonopotassium phosphate, 0.5 g/L sodium chloride, and 20 g/L dextrose)were inoculated from a 3 mL overnight culture of YPD (1% Yeast Extract,2% Peptone, 2% Dextrose), and used for RNA preparation. Cultures wereharvested by centrifugation. Each pellet was resuspended in 100 mL ofYNB-Salts medium with no glucose. A 1 mL aliquot was taken for RNAisolation as a time=0 control. To each 100 mL suspension, a differentinducer was added, 1% glucose, 1% hexane or 0.05% hexanoic acid andaliquoted as two 50 mL portions into 250 mL baffled flasks and incubatedfor 2 or 4 hours at 30° C. with shaking. At 2 hours and 4 hours, oneflask for each inducer was harvested by centrifugation and resuspendedin its own spent media in order to collapse culture foam. 1 mL sampleswere isolated by centrifugation of 1 mL of each culture and RNA preparedusing the RiboPure-Yeast Kit, according to the manufacturer'sdirections, with an additional extraction of the initial RNApreparations with 1 volume of Chloroform:lsoamyl Alcohol (24:1) to theaqueous phase after lysis and extraction with Phenol:Chloroform:lsoamylAlcohol (25:24:1).

Each RNA preparation was further purified by precipitation with ethanoland treatment with DNase I, again according to manufacturers'recommendations. All RNA preparations were shown to be free ofcontaminating genomic DNA by electrophoresis and by failure to prime aPCR product of the URA3 gene. First strand synthesis reactions werecompleted for each RNA preparation using Superscript III ReverseTranscriptase (Invitrogen), as described herein. Reactions for eachsample consisted of 1 uL oAA0542 (polyT 10 uM), 1 uL dNTP mix (10 mMeach), 1 μg RNA in 13 uL sterile, distilled water. The RNA/primer mixwas heated to 65° C. for 5 minutes, and on ice for 1 minute. Primerswere generated that amplified a substantially unique area of eachcytochrome P450 and are shown in the table below.

PCR reactions were performed on the 2 hour induced cDNA samples andcompared to the Time=0 and genomic DNA controls. PCR reactions for eachcDNA and primer pair combination consisted of 0.5 uL template, sense andantisense primers at 0.4 uM each, 1× Taq DNA polymerase Buffer (NewEngland Biolabs), 0.1 uL Taq DNA polymerase, and 0.2 mM dNTPs andsterile, distilled water to 25 uL. Cycling parameters used were 95° C.for 5 minutes, 30 cycles of 95° C. 30 seconds, 50° C. 40 seconds, 72° C.2 minutes, followed by 72° C. 5 minutes and a 4° C. hold. PCR reactionswere electrophoresed on 1.2% agarose gels to identify differentialexpression due to the inducer used. Several P450s displayed increasedinduction in the presence of hexane or hexanoic acid, however theresults were not quantitative. Two Cytochrome P450's, CYP52A15 andCYP52A16, showed induction only in the presence of hexane and hexanoicacid and not in the presence of glucose, as shown in FIG. 13. Theprimers used for PCR analysis of induced expression are shown in thetable below.

Oligonucleotides for identification of P450 DNA fragments P450 PCRproduct Oligos Sequence P450 sequence (bp) oAA0082gattactgcagcagtattagtcttc CYP52A12 60-249 190 oAA0083gtcgaaaacttcatcggcaaag oAA0084 cacgatattatcgccacatacttc CYP52A13 10-256247 oAA0085 cgggacgatcgagatcgtggatacg oAA0086 caggatattatcgccacatacatcCYP52A14 10-256 247 oAA0087 ctggacgattgagcgcttggatacg oAA0088cgtcttctccatcgtttgcccaagag CYP52A15  5-199 195 oAA0089ggtccctgacaaagttaccgagtg oAA0090 cgtcttctccatcgtttgctcaggag CYP52A16 5-199 195 oAA0091 gatccaacacgacgttaccgagcg oAA0092ggtatgtcgttgtgccagtgttg CYP52A17 26-248 223 oAA0093cccacgcttgggttcttggagtggtc oAA0094 ggtatattgttgtgcctgtgttg CYP52A1826-248 223 oAA0095 ccgacgcttgggttcttggagctgtc oAA0096ggaaggatgaggtggtgcagtac CYP52A19 1217-1458  242 oAA0097gtcttgtgacaagtttggaaactc oAA0098 gaaagaatgaggtggtgcaatac CYP52A201217-1458  242 oAA0099 gtcctgtgacaagctagggaattc oAA0104ctatcgtgggatgtgatctgtgtcg CYP52D2 19-231 213 oAA0105ctcgaatctcttgacactgaactcg

Example 18 Cloning and Analysis of C. tropicalis Fatty Alcohol Oxidase(FAO) Alleles

Isolation of Fatty Alcohol Oxidase Genes from C. tropicalis

C. tropicalis (ATCC20336) fatty alcohol oxidase genes were isolated byPCR amplification using primers generated to amplify the sequence regioncovering promoter, fatty alcohol oxidase gene (FAO) and terminator ofthe FAO1 sequence (GenBank accession number of FAO1 AY538780). Theprimers used to amplify the fatty alcohol oxidase nucleotide sequencesfrom C. tropicalis strain ATCC20336, are showing in the table below.

Oligonucleotides for cloning FAO alleles Oligo Sequence oAA0144AACGACAAGATTAGATTGGTTGAGA oAA0145 GTCGAGTTTGAAGTGTGTGTCTAAG oAA0268AGATCTCATATGGCTCCATTTTTGCCCGACCAGGTCGACT ACAAACACGTC oAA0269ATCTGGATCCTCATTACTACAACTTGGCTTTGGTCTTCAAGG AGTCTGCCAAACCTAAC oAA0282ACATCTGGATCCTCATTACTACAACTTGGCCTTGGTCT oAA0421CACACAGCTCTTCTAGAATGGCTCCATTTTTGCCCGACCAGG TCGAC oAA0422CACACAGCTCTTCCTTTCTACAACTTGGCTTTGGTCTTCAAG GAGTCTGC oAA0429GTCTACTGATTCCCCTTTGTC oAA0281 TTCTCGTTGTACCCGTCGCA

PCR reactions contained 25 uL 2× master mix, 1.5 uL of oAA0144 andoAA0145 (10 uM), 3.0 uL genomic DNA, and 19 uL sterile H₂O.Thermocycling parameters used were 98° C. for 2 minutes, 35 cycles of98° C. 20 seconds, 52° C. 20 seconds, 72° C. 1 minute, followed by 72°C. 5 minutes and a 4° C. hold. PCR products of the correct size were gelpurified, ligated into pCR-Blunt II-TOPO (Invitrogen) and transformedinto competent TOP10 E. coli cells (Invitrogen). Clones containing PCRinserts were sequenced to confirm correct DNA sequence. Four FAO alleleswere identified from sequence analysis and designated as FAO-13, FAO-17,FAO-18 and FAO-20. The sequence of the clone designated FAO-18 had asequence that was substantially identical to the sequence of FAO1 fromGen Bank. The resulting plasmids of the four alleles were designatedpAA083, pAA084, pAA059 and pAA085, respectively. Sequence identitycomparisons of FAO genes isolated as described herein are shown in thetables below.

DNA sequence identity FAO- FAO- FAO- FAO- FAO1 18 17 13 20 FAO2a FAO2bFAO1 100 100 98 96 95 83 82 FAO-18 100 98 96 95 83 82 FAO-17 100 98 9883 82 FAO-13 100 99 83 83 FAO-20 100 83 83 FAO2a 100 96 FAO2b 100

Protein sequence identity FAO- FAO- FAO- FAO- FAO1 18 17 13 20 FAO2aFAO2b FAO1 100 100 99 98 98 81 80 FAO-18 100 99 98 98 81 80 FAO-17 10099 99 82 81 FAO-13 100 99 82 81 FAO-20 100 82 81 FAO2a 100 97 FAO2b 100

Amino acid differences in FAO alleles 32 75 89 179 185 213 226 352 544590 FAO1 E M G L Y T R H S P FAO-13 Q T A L Y A K Q A A FAO-20 Q T A M DA K Q A A

Expression of FAO Alleles in E. coli

To determine the levels of FAO enzyme activity with respect to variouscarbon sources, the four isolated FAO alleles were further cloned andover-expressed in E. coli. The FAOs were amplified using the plasmidsmentioned above as DNA template by PCR with primers oAA0268 and oAA0269for FAO-13 and FAO-20 and oAA0268 and oAA0282 for FAO-17 and FAO-18,using conditions as described herein. PCR products of the correct sizewere gel purified and ligated into pET11a vector between NdeI and BamHIsites and transformed into BL21 (DE3) E. coli cells. The coloniescontaining corresponding FAOs were confirmed by DNA sequencing.Unmodified pET11a vector also was transformed into BL21 (DE3) cells, asa control. The resulting strains and plasmids were designated sAA153(pET11a), sAA154 (pAA079 containing FAO-13), sAA155 (pAA080 containingFAO-17), sAA156 (pAA081 containing FAO-18) and sAA157 (pAA082 containingFAO-20), respectively. The strains and plasmids were used for FAOover-expression in E. coli. One colony of each strain was transferredinto 5 mL of LB medium containing 100 μg/mL ampicillin and grownovernight at 37° C., 200 rpm. The overnight culture was used toinoculate a new culture to OD_(600nm) 0.2 in 25 ml LB containing 100μg/ml ampicillin. Cells were induced at OD_(600nm) 0.8 with 0.3 mM IPTGfor 3 hours and harvested by centrifugation at 4° C. 1,050×g for 10minutes. The cell pellet was stored at −20° C.

Expression of FAOs in C. tropicalis

Two alleles, FAO-13 and FAO-20, were chosen for amplification in C.tropicalis based on their substrate specificity profile, as determinedfrom enzyme assays of soluble cell extracts of E. coli with overexpressed FAOs. DNA fragments containing FAO-13 and FAO-20 wereamplified using plasmids pAA079 and pAA082 as DNA templates,respectively, by PCR with primers oAA0421 and oAA0422. PCR products ofthe correct sizes were gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing FAO inserts were sequenced to confirmcorrect DNA sequence. Plasmids containing FAO-13 and FAO-20 weredigested with Sapl and ligated into vector pAA105, which includes the C.tropicalis PGK promoter and terminator. The resulting plasmids wereconfirmed by restriction digestion and DNA sequencing and designated aspAA115 (FAO-13) and pAA116 (FAO-20), respectively. Plasmids pAA115 andpAA116 were linearized with SpeI, transformed into competent C.tropicalis Ura ⁻ strains sAA002 (SU-2, ATCC20913) and sAA103. Theintegration of FAO-13 and FAO-20 was confirmed by colony PCR usingprimers oAA0429 and oAA0281. The resulting strains were designated assAA278 (pAA115 integrated in strain sAA002), sAA280 (pAA116 integratedin sAA002), sAA282 (pAA115 integrated in sAA103), and sAA284 (pAA116integrated in sAA103), and were used for fatty alcohol oxidaseover-expression in C. tropicalis.

One colony of each strain was inoculated into 5 ml YPD and grownovernight as described herein. The overnight culture was used toinoculate a new 25 mL YPD culture to about OD_(600nm) 0.5. FAOover-expression was regulated by the PGK promoter/terminator, inducedwith glucose in the medium and expressed constitutively. Strains sAA002and sAA103 (e.g., untransformed starting strains) were included asnegative controls for FAO over-expression. Cells were harvested at earlylog phase (OD_(600nm)=in the range of between about 3 to about 5) bycentrifugation at 4° C. for 10 minutes at 1,050×g. Cell pellets werestored at −20° C.

Cell Extract Preparation from E. coli

Cell pellets from 25 mL of FAO expressing E. coli cultures wereresuspended in 10 mL phosphate-glycerol buffer containing 50 mMpotassium phosphate buffer (pH7.6), 20% glycerol, 1 mMPhenylmethylsulfonyl fluoride (PMSF), 2 uL Benzonase 25 U/uL, 20 uL 10mg/mL lysozyme. The cells were then lysed by incubation at roomtemperature for 50 minutes on a rotating shaker, and the cell suspensioncentrifuged for 30 minutes at 4° C. using 15,000×g for. The supernatantwas aliquoted in 1.5 ml microcentrifuge tubes and stored at −20° C. forFAO enzyme activity assays.

Cell Extract Preparation from C. tropicalis

Frozen C. tropicalis cell pellets were resuspended in 1.2 ml ofphosphate-glycerol buffer containing 50 mM potassium phosphate buffer(pH7.6), 20% glycerol, 1 mM Phenylmethylsulfonyl fluoride (PMSF).Resuspended cells were transferred to 1.5 mL screw-cap tubes containingabout 500 uL of zirconia beads on ice. The cells were lysed with a BeadBeater (Biospec) using 2 minute pulses and 1 minute rest intervals onice. The process was repeated 3 times. The whole cell extract was thentransferred to a new 1.5 ml tube and centrifuged at 16,000×g for 15minutes at 4° C. The supernatant was transferred into a new tube andused for FAO enzyme activity assays.

Protein Concentration Determination

Protein concentration of the cell extracts was determined using theBradford Reagent following manufacturers' recommendations (Cat# 23238,Thermo scientific).

FAO Enzyme Activity Assay

FAO enzyme activity assays were performed using a modification of Eirichet al., 2004). The assay utilizes a two-enzyme coupled reaction (e.g.,FAO and horse radish peroxidase (HRP)) and can be monitored byspectrophotometry. 1-Dodecanol was used as a standard substrate forfatty alcohol oxidase enzymatic activity assays. FAO oxidizes thedodecanol to dodecanal while reducing molecular oxygen to hydrogenperoxide simultaneously. HRP reduces (2,2′-azino-bis3-ethylbenzothiazoline-6-sulfonic acid; ABTS) in the two-enzyme coupledreaction, where the electron obtained from oxidizing hydrogen peroxideto ABTS, which can be measured by spectrometry at 405 nm. The assay wasmodified using aminotriazole (AT) to prevent the destruction of H₂O₂ byendogenous catalase, thus eliminating the need for microsomalfractionation. The final reaction mixture (1.0 mL) for FAO enzyme assayconsisted of 500 μL of 200 mM HEPES buffer, pH 7.6; 50 μL of a 10 mg/mLABTS solution in deionized water; 10 μL of 5 mM solution of dodecanol inacetone; 40 μL of 1M AT and 5 μL of a 2 mg/mL horseradish peroxidasesolution in 50 mM potassium phosphate buffer, pH 7.6. Reaction activitywas measured by measuring light absorbance at 405 nm for 10 minutes atroom temperature after adding the extract. The amount of extract addedto the reaction mixture was varied so that the activity fell within therange of 0.2 to 1.0 ΔA_(405nm)/min. The actual amounts of extract usedwere about 1.69 U/mg for E. coli expressed FAO-13, 0.018 U/mg for E.coli expressed FAO-17, 0.35 U/mg for E. coli expressed FAO-18 (e.g.,FAO1), 0.47 U/mg E. coli expressed FAO-20, 0.036 U/mg C. tropicalis(strain sAA278) expressed FAO-13, 0.016 U/mg C. tropicalis (strainsAA282) expressed FAO-13, 0.032 U/mg C. tropicalis (strain sAA280)expressed FAO-20 and 0.029 U/mg C. tropicalis (strain sAA284) expressedFAO-20. FAO activity was reported as activity units/mg of total protein(1 unit=1 mmole substrate oxidized/min). An extinction coefficient at405 nm of 18.4 was used for ABTS and was equivalent to 0.5 mM oxidizedsubstrate. The results of the activity assays are shown in the tablesbelow.

FAO activity (units/mg total protein) on primary alcohols 1- 1- 1- 1- 1-1- 1- Butanol Pentanol Hexanol Octanol Decanol Dodecanol TetradecanolHexadecanol FAO-13 0.01 0.09 1.17 82.67 70.94 100 79.35 58.88 FAO-170.72 0.26 1.06 66.23 22.00 100 47.86 60.98 FAO-18 0.07 0.11 0.26 60.5654.56 100 114.47 50.65 FAO-20 0.07 0.11 0.91 55.96 74.57 100 89.52 42.59

FAO activity (units/mg total protein) on omega hydroxy fatty acids 1-12-OH- 16-OH- Dodecanol 6-OH-HA 10-OH-DA DDA HDA FAO-13 100 4.18 4.146.87 8.57 FAO-17 100 1.18 0.00 0.59 0.94 FAO-18 100 0.00 0.00 4.87 2.94FAO-20 100 0.03 0.04 2.25 7.46

Example 19 Construction of C. tropicalis Shuttle Vector pAA061

Vector pAA061 was constructed from a pUC19 backbone to harbor theselectable marker URA3 from C. tropicalis strain ATCC20336 as well asmodifications to allow insertion of C. tropicalis promoters andterminators. A 1,507 bp DNA fragment containing the promoter, ORF, andterminator of URA3 from C. tropicalis ATCC20336 was amplified usingprimers oAA0124 and oAA0125, shown in the table below. The URA3 PCRproduct was digested with NdeI/MluI and ligated into the 2,505 bpfragment of pUC19 digested with NdeI/BsmBI (an MluI compatible overhangwas produced by BsmBI). In order to replace the Iac promoter with ashort 21 bp linker sequence, the resulting plasmid was digested withSphI/SapI and filled in with a linker produced by annealing oligosoAA0173 and oAA0174. The resulting plasmid was named pAA061, and isshown in FIG. 30.

Oligonucleotides for construction of pAA061 PCR product Oligos Sequence(bp) oAA0124 cacacacatatgCGACGGGTACAACGAGAATT 1507 oAA0125cacacaacgcgtAGACGAAGCCGTTCTTCAAG oAA0173 ATGATCTGCCATGCCGAACTC 21oAA0174 AGCGAGTTCGGCATGGCAGATCATCATG (linker)

Example 20 Cloning of C. tropicalis PGK Promoter and Terminator

Vector pAA105 was constructed from base vector pAA061 to include thephosphoglycerate kinase (PGK) promoter and terminator regions from C.tropicalis ATCC20336 with an intervening multiple cloning site (MCS) forinsertion of open reading frames (ORF's). The PGK promoter region wasamplified by PCR using primers oAA0347 and oAA0348, shown in the tablebelow. The 1,029 bp DNA fragment containing the PGK promoter wasdigested with restriction enzymes PstI/XmaI. The PGK terminator regionwas amplified by PCR using primers oAA0351 and oAA0352, also shown inthe table below. The 396 bp DNA fragment containing the PGK terminatorwas digested with restriction enzymes XmaI/EcoRI. The 3,728 bpPstI/EcoRI DNA fragment from pAA061 was used in a three piece ligationreaction with the PGK promoter and terminator regions to produce pAA105.The sequence between the PGK promoter and terminator containsrestriction sites for incorporating ORF's to be controlled by thefunctionally linked constitutive PGK promoter.

Oligonucleotides for cloning C. tropicalis PGK promoter and terminatorPCR product Oligos Sequence (bp) oAA0347CACACACTGCAGTTGTCCAATGTAATAATTTT 1028 oAA0348CACACATCTAGACCCGGGCTCTTCTTCTGAA TAGGCAATTGATAAACTTACTTATC oAA0351GAGCCCGGGTCTAGATGTGTGCTCTTCCAA 396 AGTACGGTGTTGTTGACA oAA0352CACACACATATGAATTCTGTACTGGTAGAG CTAAATT

Example 21 Cloning of the POX4 Locus

Primers oAA0138 and oAA0141 (shown in the table below) were generated toamplify the entire sequence of NCBI accession number M12160 for theYSAPOX4 locus from genomic DNA prepared from C. tropicalis strainATCC20336. The 2,845 bp PCR product was cloned into the vector,pCR-BluntII-TOPO (Invitrogen), sequenced and designated pAA052, and isshown in FIG. 29.

Oligonucleotides for cloning of POX4 Oligos Sequence PCR product (bp)oAA0138 GAGCTCCAATTGTAATATTTCGGG 2845 oAA0141 GTCGACCTAAATTCGCAACTATCAA

Example 22 Cloning of the POX5 Locus

Primers oAA0179 and oAA0182 (shown in the table below) were generated toamplify the entire sequence of NCBI accession number M12161 for theYSAPOX5 locus from genomic DNA prepared from C. tropicalis strainATCC20336. The 2,624 bp PCR product was cloned into the vector,pCR-BluntII-TOPO (Invitrogen), sequenced and designated pAA049, and isshown in FIG. 28.

Oligonucleotides for cloning of POX5 PCR product Oligos Sequence (bp)oAA0179 GAATTCACATGGCTAATTTGGCCTCGGTTCCACA 2624 ACGCACTCAGCATTAAAAAoAA0182 GAGCTCCCCTGCAAACAGGGAAACACTTGTCAT CTGATTT

Example 23 Construction of Strain sAA105 and sAA106

Functional POX4 alleles were restored in C. tropicalis strain sAA003(ATCC20962; ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/pox5::URA3) bytransformation of sAA003 with POX4 linear DNA to replace theura3-disrupted loci with functional alleles. A 2,845 bp DNA fragment wasamplified by PCR using primers oAA0138 and oAA0141 (described in Example21) that contained the POX4 ORF as well as 531 bp upstream and 184 bpdownstream of the ORF, using plasmid pAA052 as template. The purifiedPCR product was used to transform competent sAA003 cells which wereplated on YNB-agar plates supplemented with hexadecane vapor as thecarbon source (e.g., by placing a filter paper soaked with hexadecane inthe lid of the inverted petri dish) and incubated at 30° C. for 4-5days. Colonies growing on hexadecane as the sole carbon source wererestreaked onto YPD-agar and incubated at 30° C. Single colonies weregrown in YPD cultures and used for the preparation of genomic DNA.

PCR analysis of the genomic DNA prepared from the transformants wasperformed with oligos oAA0138 and oAA0141. An URA3-disrupted POX4 wouldproduce a PCR product of 5,045 bp, while a functional POX4 would producea PCR product of 2,845 bp. In strain sAA105 only one PCR product wasamplified with a size of 2,845 bp indicating that both POX4 alleles hadbeen functionally restored. In strain sAA106 PCR products of both 2,845bp and 5,045 bp were amplified indicating that one POX4 allele had beenfunctionally restored while the other POX4 allele remained disrupted byURA3. The resultant strain genotypes were: sAA105 (ura3/ura3, POX4/POX4,pox5::ura3/pox5::URA3) and sAA106 (ura3/ura3, POX4/pox4::ura3,pox5::ura3/pox5::URA3).

Example 24 Construction of Strain sAA152

Functional POX5 alleles were restored in C. tropicalis strain sAA103(ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3) bytransformation of sAA103 with PmII-linearized plasmid pAA086 (containingthe POX5 promoter, gene, terminator and a URA3 marker). Selection oftransformants was performed by plating on SC-URA agar plates.Verification of plasmid integration was performed by PCR with primersoAA179 and oAA182 (described in Example 22). Integration of the plasmidwas shown by a PCR product of 2,584 bp indicating the presence of afunctional POX5 allele. Other POX5 alleles in sAA152 were disrupted withan ura3 gene increasing the PCR product size for nonfunctional allelesto 4,734 bp. Genotype for strain sAA152 is ura3/ura3,pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3, ura3::POX5,URA3).

Example 25 Construction of Strain sAA232

Functional POX5 alleles were restored in C. tropicalis strain sAA003 bytransformation of sAA003 with POX5 linear DNA to replace theURA3-disrupted loci with a functional allele. A 2,584 bp DNA fragmentwas amplified by PCR using primers oAA0179 and oAA0182 (described inExample 22) that contained the POX5 ORF as well as 456 bp upstream and179 bp downstream of the ORF using plasmid pAA049 as template. Thepurified PCR product was used to transform competent sAA003 cells whichwere plated on SC+URA+5FOA plates and incubated at 30° C. for 3-4 days.Colonies were restreaked onto YPD-agar and incubated at 30° C. Singlecolonies were grown in YPD cultures and used for the preparation ofgenomic DNA. PCR analysis of the genomic DNA prepared from thetransformants was performed with oligos oAA0179 and oAA0182. Anura3-disrupted POX5 would produce a PCR product of 4,784 bp while afunctional POX5 would produce a PCR product of 2,584 bp. In strainsAA232 PCR products of both 2,584 bp and 4,784 bp were amplifiedindicating that one POX5 allele had been functionally restored while theother POX5 allele remained disrupted by ura3. The resultant genotype ofstrain sAA232 is ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/POX5.5-FOA selection restored the POX5 allele that had been disrupted withthe functional URA3 leaving the sAA232 strain Ura⁻.

Example 26 Construction of Strain sAA235

Functional POX5 alleles were restored in C. tropicalis strain sAA003 bytransformation of sAA003 with POX5 linear DNA to replace theURA3-disrupted loci with a functional allele. A 2,584 bp DNA fragmentwas amplified by PCR using primers oAA0179 and oAA0182 (described inExample 22) that contained the POX5 ORF as well as 456 bp upstream and179 bp downstream of the ORF using plasmid pAA049 as template. Thepurified PCR product was used to transform competent sAA003 cells whichwere plated on YNB-agar plates supplemented with dodecane vapor as thecarbon source (e.g., by placing a filter paper soaked with dodecane inthe lid of the inverted petri dish) and incubated at 30° C. for 4-5days. Colonies growing on dodecane as the sole carbon source wererestreaked onto YPD-agar and incubated at 30° C. Single colonies weregrown in YPD cultures and used for the preparation of genomic DNA. PCRanalysis of the genomic DNA prepared from the transformants wasperformed with oligos oAA0179 and oAA0182. An ura3-disrupted POX5 wouldproduce a PCR product of 4,784 bp while a functional POX5 would producea PCR product of 2,584 bp. In strain sAA235 a PCR product of 2,584 bpwas amplified indicating that both POX5 alleles had been functionallyrestored. An unintended consequence of the selection strategy (YNB-agarwith dodecane) was that the cells reverted back to an Ura⁺ phenotype.Without being limited by any theory, it is believed the absence ofuracil in the solid media and the replacement of the only functionalURA3 forced the cells to mutate one of the other ura3 loci back to afunctional allele. Therefore the genotype of the strain sAA235 isbelieved to be URA3/ura3, pox4::ura3/pox4::ura3, POX5/POX5. Verificationof which of the loci is the functional URA3 is underway.

Example 27 Construction of Strains with Amplified CPR and CYP52 Genes

Strains having an increased number of copies of cytochrome P450reductase (CPR) and/or for cytochrome P450 monooxygenase (CYP52) geneswere constructed to determine how over expression of CPR and CYP52affected diacid production.

Cloning and Integration of the CPR Gene.

A 3,019 bp DNA fragment encoding the CPR promoter, ORF, and terminatorfrom C. tropicalis ATCC750 was amplified by PCR using primers oAA0171and oAA0172 (see table below) incorporating unique SapI and SphI sites.The amplified DNA fragment was cut with the indicated restrictionenzymes and ligated into plasmid pAA061, shown in FIG. 30, to produceplasmid pAA067, shown in FIG. 32. Plasmid pAA067 was linearized withClaI and transformed into C. tropicalis Ura⁻ strain sAA103 (ura3/ura3,pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). Transformations wereperformed with plasmid pAA067 alone and in combination with plasmidsharboring the CYP52A15 or CYP52A16 genes, described below.

Cloning and Integration of CYP52A15 Gene.

A 2,842 bp DNA fragment encoding the CYP52A15 promoter, ORF, andterminator from C. tropicalis ATCC20336 was amplified by PCR usingprimers oAA0175 and oAA0178 (see table below) and cloned intopCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A15 DNAfragment was isolated by restriction digest with XbaI/BamHI (2,742 bp)and ligated into plasmid pAA061, shown in FIG. 30, to produce plasmidpAA077, shown in FIG. 33. Plasmid pAA077 was linearized with PmII andtransformed into C. tropicalis Ura⁻ strain sAA103 (ura3/ura3,pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). pAA077 was cotransformedwith plasmid pAA067 harboring the CPR gene.

Cloning and Integration of CYP52A16 Gene.

A 2,728 bp DNA fragment encoding the CYP52A16 promoter, ORF, andterminator from C. tropicalis ATCC20336 was amplified by PCR usingprimers oAA0177 and oAA0178 (see table below) and cloned intopCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A16 DNAfragment was amplified with primers oAA0260 and oAA0261 (see tablebelow) which incorporated unique SacI/XbaI restriction sites. Theamplified DNA fragment was digested with SacI and XbaI restrictionenzymes and ligated into plasmid pAA061 to produce plasmid pAA078, shownin FIG. 34. Plasmid pAA078 was linearized with ClaI and transformed intoC. tropicalis Ura⁻ strain sAA103 (ura3/ura3, pox4::ura3/pox4::ura3,pox5::ura3/pox5::ura3). pAA078 was cotransformed with plasmid pAA067harboring the CPR gene.

Oligonucleotides for cloning of CPR, CYP52A15 and CYP52A16 PCR productOligos Sequence (bp) oAA0171 cacctcgctcttccAGCTGTCATGTCTATTCAAT 3019GCTTCGA oAA0172 cacacagcatgcTAATGTTTATATCGTTGACG GTGAAA oAA0175cacaaagcggaagagcAAATTTTGTATTCTCAGT 2842 AGGATTTCATC oAA0178cacacagcatgCAAACTTAAGGGTGTTGTAGATA TCCC oAA0177cacacacccgggATCGACAGTCGATTACGTAATC 2772 CATATTATTT oAA0178cacacagcatgCAAACTTAAGGGTGTTGTAGAT ATCCC oAA0260cacacagagctcACAGTCGATTACGTAATCCAT 2772 oAA0261cacatctagaGCATGCAAACTTAAGGGTGTTGTA

Preparation of Genomic DNA.

Genomic DNA was prepared from transformants for PCR verification and forSouthern blot analysis. Isolated colonies were inoculated into 3 mL YPDand grown overnight at 30° C. with shaking. Cells were pelleted bycentrifugation. To each pellet, 200 uL Breaking Buffer (2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris pH 8 and, 1 mM EDTA) was added, and thepellet resuspended and transferred to a fresh tube containing 200 uL 0.5mm Zirconia/Silica Beads. 200 uL Phenol:Chloroform:lsoamyl Alcohol(25:24:1) was added to each tube, followed by vortexing for 1 minute.Sterile distilled water was added (200 uL) to each tube and the tubeswere centrifuged at 13000 rpm for 10 minutes. The aqueous layer wasethanol precipitated and washed with 70% ethanol. The pellet wasresuspended in 100-200 μl 10 mM Tris, after drying. Genomic DNApreparation for southern blot analysis was performed using the sameprocedure on 25 mL cultures for each colony tested.

Characterization of Strains with Amplified CPR and CYP52 Genes.

Verification of integrants was performed by PCR using primers oAA0252and oAA0256 (CPR), oAA0231 and oAA0281 (CYP52A15), and oAA242 andoAA0257 (CYP52A16). The primers used for verification are shown in thetable below.

Oligonucleotides for PCR verification of CPR, CYP52A15 and CYP52A16Oligos Sequence PCR product (bp) oAA0252 TTAATGCCTTCTCAAGACAA 743oAA0256 GGTTTTCCCAGTCACGACGT oAA0231 CCTTGCTAATTTTCTTCTGTATAGC 584oAA0281 TTCTCGTTGTACCCGTCGCA oAA0242 CACACAACTTCAGAGTTGCC 974 oAA0257TCGCCACCTCTGACTTGAGC

Southern blot analysis was used to determine the copy number of the CPR,CYP52A15 and CYP52A15 genes. Biotinylated DNA probes were prepared withgene specific oligonucleotides using the NEBlot Phototope Kit from NewEngland BioLabs (Catalog #N7550S) on PCR products generated from eachgene target as specified in the table below. Southern Hybridizationswere performed using standard methods (e.g., Sambrook, J. and Russell,D. W. (2001) Molecular Cloning: A Laboratory Manual, (3^(rd) ed.), pp.6.33-6.64. Cold Spring Harbor Laboratory Press). Detection of hybridizedprobe was performed using the Phototope-Star Detection Kit from NewEngland BioLabs (Catalog #N7020S). Copy number was determined bydensitometry of the resulting bands.

Oligonucleotides for Probe Template PCR of CPR, CYP52A15 and CYP52A16PCR product Oligos Sequence Gene Template (bp) oAA0250 AATTGAACATCAGAAGACPR pAA067 1313 GGA oAA0254 CCTGAAATTTCCAAATGG TGTCTAA oAA0227TTTTTTGTGCGCAAGTA CYP52A15 pAA077 905 CAC oAA0235 CAACTTGACGTGAGAAAC CToAA0239 AGATGCTCGTTTTACACC CYP52A16 pAA078 672 CT oAA0247ACACAGCTTTGATGTTCT CT

Example 28 Strain Evaluation of Partially β-Oxidation Blocked Strains

Fermentation of Methyl Laurate Feedstock.

5 mL starter cultures, in SP92 media (6.7 g/L Difco yeast nitrogen base,3.0 g/L Difco yeast extract, 3.0 g/L ammonium sulfate, 1.0 g/L potassiumphosphate monobasic, 1.0 g/L potassium phosphate dibasic, 75 g/Ldextrose) were incubated overnight at 30° C., with shaking and used toinoculate flasks containing 25 mL of SP92 media to an initial OD_(600nm)of about 0.4. Cultures were incubated approximately 18 hours at 30° C.,with shaking at about 200 rpm. Cells were pelleted by centrifugation at4° C. for 10 minutes at 4,000×g, then resuspended in SP92-D media (6.7g/L Difco yeast nitrogen base, 3.0 g/L Difco yeast extract, 3.0 g/Lammonium sulfate, 1.0 g/L potassium phosphate monobasic, 1.0 g/Lpotassium phosphate dibasic) supplemented with 0.1% dextrose and 2%methyl laurate. Incubation of the cultures continued at 30° C., withshaking and samples were taken for analysis of fatty acids and diacidsby gas chromatography (GC).

Sample for GC were prepared by adding 0.8 mL of 6.0M HCl to 1 mL ofwhole culture samples and the samples were stored at 4° C. to awaitprocessing. Samples were processed by incubating in a 60° C. water bathfor 5 minutes, after which 4.0 mL of MTBE was added to the 1.8 mLacidified whole culture samples and vortexed for 20 seconds. The phaseswere allowed to separate for 10 min at room temperature. 1 mL of theMTBE phase was drawn and dried with sodium sulfate. Aliquots of the MTBEphase were derivatized with BSTFA reagent (Regis Technologies Inc.) andanalyzed by GC equipped with a Flame Ionization Detector. The results ofthe gas chromatography are shown in the table below.

Fatty acid and Diacid profile (g/L) in Methyl Laurate fermentation TimeC12 C12 C10 C8 C6 Strain (h) Acid Diacid Diacid Diacid Diacid sAA105 00.00 0.00 0.00 0.00 0.00 24 0.05 0.00 0.00 0.07 0.42 48 0.00 0.00 0.000.00 0.00 sAA106 0 0.00 0.00 0.00 0.00 0.00 24 2.92 1.29 0.15 0.58 0.3748 0.04 0.02 0.00 0.00 0.01 sAA152 0 0.02 0.00 0.00 0.00 0.00 24 0.580.55 0.07 0.43 0.03 48 0.00 0.03 0.00 0.05 0.58 sAA003 0 0.00 0.00 0.000.00 0.00 24 1.96 0.41 0.00 0.00 0.00 48 1.43 0.47 0.00 0.00 0.00

Fermentation of Methyl Myristate and Oleic Acid Feedstocks.

Fermentations were performed essentially as described for methyl lauratefeedstock except that 2% methyl myristate or 2% oleic acid wassubstituted for the 2% methyl laurate. The results of the gaschromatography are shown in the tables below.

Fatty acid and Diacid profile (g/L) in Methyl Myristate fermentationTime C14 C14 C12 C10 C8 C6 Strain (h) Acid Diacid Diacid Diacid DiacidDiacid sAA105 0 0.02 0.00 0.00 0.00 0.00 0.00 24 0.02 0.00 0.00 0.000.00 0.29 48 0.01 0.00 0.00 0.00 0.00 0.03 sAA106 0 0.01 0.00 0.00 0.000.00 0.00 24 0.02 0.00 0.00 0.00 0.08 1.71 48 0.01 0.00 0.00 0.00 0.000.04 sAA232 0 0.01 0.00 0.00 0.00 0.00 0.00 24 0.01 0.00 0.00 0.00 0.590.26 48 0.01 0.00 0.00 0.00 0.35 0.47 sAA235 0 0.01 0.00 0.00 0.00 0.000.00 24 0.02 0.00 0.00 0.00 0.25 0.38 48 0.01 0.00 0.00 0.00 0.04 0.66sAA003 0 0.02 0.01 0.01 0.00 0.00 0.00 24 0.55 0.25 0.00 0.00 0.00 0.0048 0.49 0.38 0.00 0.00 0.00 0.00

Diacid profile (g/L) in Oleic acid fermentation Time C14 C12 C10 C8 C6Strain (h) Diacid Diacid Diacid Diacid Diacid sAA105 0 0.00 0.00 0.000.00 0.00 24 0.00 0.00 0.00 0.00 0.03 48 0.00 0.00 0.00 0.00 0.01 sAA1060 0.00 0.00 0.00 0.00 0.00 24 0.00 0.00 0.00 0.00 1.48 48 0.00 0.00 0.000.00 0.42 sAA232 0 0.00 0.00 0.00 0.00 0.00 24 0.00 0.00 0.00 0.00 0.0948 0.00 0.00 0.00 0.00 0.10 sAA235 0 0.00 0.00 0.00 0.00 0.00 24 0.000.00 0.00 0.00 0.10 48 0.00 0.00 0.00 0.00 0.11

Fermentations also were performed using coconut oil as a feed stock.Coconut oil contains a mixture of fatty acids of different carbon chainlengths. The percent composition of fatty acids, by weight, is about 6%capric acid (C10:0, where 0 refers to the number of double orunsaturated bonds), about 47% lauric acid (C12:0), about 18% myristicacid (C14:0), about 9% palmitic acid (C16:0). About 3% stearic acid(C18:0), about 6% oleic acid (C18:1, where 1 refers to the number ofdouble bonds), and about 2% linoleic acid (omega-6 fatty acid, C18:2).In some embodiments, palm kernel oil can be substituted for coconut oil.Palm kernel oil has a distribution of fatty acids similar to that ofcoconut oil. Fermentations and GC were carried out essentially asdescribed herein with the exception of feedstock used. The result offermentations performed using coconut oil as a feedstock are presentedbelow.

Diacid profile (g/L) in Coconut Oil fermentation Time C14 C12 C10 C8 C6Strain (h) Diacid Diacid Diacid Diacid Diacid sAA105 0 0.00 0.00 0.000.00 0.00 24 0.00 0.00 0.00 0.00 0.00 48 0.00 0.00 0.00 0.00 0.00 sAA1060 0.00 0.00 0.00 0.00 0.00 24 0.00 0.00 0.00 0.00 0.01 48 0.00 0.00 0.000.00 0.00 sAA152 0 0.00 0.00 0.00 0.00 0.00 24 0.00 0.00 0.00 0.00 0.4348 0.00 0.00 0.00 0.00 0.45 sAA232 0 0.00 0.00 0.00 0.00 0.00 24 0.000.00 0.00 0.00 0.41 48 0.00 0.00 0.00 0.00 0.58 sAA235 0 0.00 0.00 0.000.00 0.00 24 0.00 0.00 0.00 0.00 0.43 48 0.00 0.00 0.00 0.00 0.76

Example 29 Strain Evaluation of Completely β-Oxidation Blocked Strains

Fermentations also were performed using methyl myristate as a feedstock. Fermentations and

GC were carried out essentially as described herein with the exceptionof feedstock used. The result of fermentations performed using coconutoil as a feedstock are presented below.

C14 Diacid production in strains with amplified CPR, CYP52A15, and/orCYP52A16 Strain C14 diacid, 72 h (g/L) CPR A15 A16 sAA003 0.98 2 1 1sAA318 1.19 3 1 1 sAA239 2.75 3 1 3 sAA319 1.37 7 1 1 sAA238 1.93 7 2 1

Example 30 Nucleic Acid and Amino Acid Sequences of Novel Fatty AlcoholOxidase Genes

As noted above, novel fatty alcohol oxidase genes were identified andcloned. The nucleotide and amino acid sequences of the novel sequencesare presented herein. Nucleotide and amino acid sequence identitycomparison are shown in Example 18.

Nucleotide Sequences FAO-13 (SEQ ID NO: 1)atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaagtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag FAO-17 (SEQ ID NO: 2)atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccgacaaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatcttgaccaatgttttgggatcaagggtcttggcaccagctttgaccaactcgttgactcctatcaaggacatgagcttggaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgctaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtttagagacgcagctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaagaagggcatcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggccccaaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattcaagaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgcaagacatgttgcgttaggtttggcagactccttgaagaccaaggccaagttgtag FAO-20 (SEQ ID NO: 3)atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaagtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaaattgttcaggttggtttccacgcttaccttggttactttcacgagattggccaatgagttgcatttgaaagccattcactatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgaccctttcaagtaccagtttatggaaaagccaaagtttgacggcgctgagttgtacttgccagatattgatgttatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag FAO2a (SEQ ID NO: 4)atgaataccttcttgccagacgtgctcgaatacaaacacgtcgacacccttttgttattgtgtgacgggatcatccacgaaaccacagtcgatcagatcaaggacgccattgctcccgacttccctgaggaccagtacgaggagtatctcaagaccttcaccaagccatctgagacccctgggttcagagaagccgtctacgacacgatcaacgccaccccaaccgatgccgtgcacatgtgtattgtcttgaccaccgcattggactccagaatcttggcccccacgttgaccaactcgttgacgcctatcaaggatatgaccttgaaggagcgtgaacaattgttggcctcttggcgtgattccccgattgcggcaaagagaagattgttcagattgatttcctcgcttaccttgacgacgtttacgagattggccagcgaattgcacttgaaagccatccactaccctggcagagacttgcgtgaaaaggcgtatgaaacccaggtggttgaccctttcaggtacctgtttatggagaaaccaaagtttgacggcgccgaattgtacttgccagatatcgacgtcatcatcattggatcaggcgccggtgctggtgtcatggcccacactctcgccaacgacgggttcaagaccttggttttggaaaagggaaagtatttcagcaactccgagttgaactttaatgacgctgatggcgtgaaagagttgtaccaaggtaaaggtgctttggccaccaccaatcagcagatgtttattcttgccggttccactttgggcggtggtaccactgtcaactggtctgcttgccttaaaacaccatttaaagtgcgtaaggagtggtacgacgagtttggtcttgaatttgctgccgatgaagcctacgacaaagcgcaggattatgtttggaaacaaatgggtgcttcaacagatggaatcactcactccttggccaacgaagttgtggttgaaggaggtaagaagttgggctacaagagcaaggaaattgagcagaacaacggtggccaccctgaccacccatgtggtttctgttacttgggctgtaagtacggtattaaacagggttctgtgaataactggtttagagacgcagctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaatcctcaacaagaatggcgtcgcttatggtatcttgtgtgaggatgtcgaaaccggagtcaggttcactattagtggccccaaaaagtttgttgtttctgctggttctttgaacacgccaactgtgttgaccaactccggattcaagaacaagcacattggtaagaacttgacgttgcacccagtttccaccgtgtttggtgactttggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctttgttacgaggttgctgacttggacggcaagggccacggatgcagaatcgaaaccatcttgaacgctccattcatccaagcttctttgttgccatggagaggaagtgacgaggtcagaagagacttgttgcgttacaacaacatggtggccatgttgcttatcacgcgtgataccaccagtggttcagtttctgctgacccaaagaagcccgacgctttgattgtcgactatgagattaacaagtttgacaagaatgccatcttgcaagctttcttgatcacttccgacatgttgtacattgaaggtgccaagagaatcctcagtccacagccatgggtgccaatctttgagtcgaacaagccaaaggagcaaagaacgatcaaggacaaggactatgttgagtggagagccaaggctgctaagatacctttcgacacctacggttctgcatatgggtccgcacatcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggtgctgttgatactgatggtagattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcctactgccagcggtgccaacccaatgatatccaccatgacctttgctagacagattgcgttaggtttggctgactccttgaagaccaaacccaagttgtag FAO2b (SEQ ID NO: 5)atgaataccttcttgccagacgtgctcgaatacaaacacgtcgatacccttttgttattatgtgacgggatcatccacgaaaccacagtcgaccagatcagggacgccattgctcccgacttccctgaagaccagtacgaggagtatctcaagaccttcaccaagccatctgagacccctgggttcagagaagccgtctacgacacgatcaacagcaccccaaccgaggctgtgcacatgtgtattgtattgaccaccgcattggactcgagaatcttggcccccacgttgaccaactcgttgacgcctatcaaggatatgaccttgaaagagcgtgaacaattgttggctgcctggcgtgattccccgatcgcggccaagagaagattgttcagattgatttcctcacttaccttgacgacctttacgagattggccagcgacttgcacttgagagccatccactaccctggcagagacttgcgtgaaaaggcatatgaaacccaggtggttgaccctttcaggtacctgtttatggaaaaaccaaagtttgacggcaccgagttgtacttgccagatatcgacgtcatcatcattggatccggtgccggtgctggtgtcatggcccacactttagccaacgacgggtacaagaccttggttttggaaaagggaaagtatttcagcaactccgagttgaactttaatgatgccgatggtatgaaagagttgtaccaaggtaaatgtgcgttgaccaccacgaaccagcagatgtttattcttgccggttccactttgggcggtggtaccactgttaactggtctgcttgtcttaaaacaccatttaaagtgcgtaaggagtggtacgacgagtttggtcttgaatttgctgccgacgaagcctacgacaaagcacaagactatgtttggaaacaaatgggcgcttctaccgaaggaatcactcactctttggcgaacgcggttgtggttgaaggaggtaagaagttgggttacaagagcaaggaaatcgagcagaacaatggtggccatcctgaccacccctgtggtttctgttacttgggctgtaagtacggtattaagcagggttctgtgaataactggtttagagacgcagctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaatcctccacaataaaggcgtcgcttatggcatcttgtgtgaggatgtcgagaccggagtcaaattcactatcagtggccccaaaaagtttgttgtttctgcaggttctttgaacacgccaacggtgttgaccaactccggattcaagaacaaacacatcggtaagaacttgacgttgcacccagtttcgaccgtgtttggtgactttggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctctgttacgaagtcgctgacttggacggcaagggccacggatgcagaatcgagaccatcttgaacgctccattcatccaagcttctttgttgccatggagaggaagcgacgaggtcagaagagacttgttgcgttacaacaacatggtggccatgttgcttatcacccgtgacaccaccagtggttcagtttctgctgacccaaagaagcccgacgctttgattgtcgactatgacatcaacaagtttgacaagaatgccatcttgcaagctttcttgatcacctccgacatgttgtacatcgaaggtgccaagagaatcctcagtccacaggcatgggtgccaatctttgagtcgaacaagccaaaggagcaaagaacaatcaaggacaaggactatgtcgaatggagagccaaggctgccaagatacctttcgacacctacggttctgcctatgggtccgcacatcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggcgccgttgataccgatggtagattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcctactgccagcggtgccaacccaatgatctccaccatgacgtttgctagacagattgcgttaggtttggctgactctttgaagaccaaacccaagttgtag

In addition to the novel FAO genes isolated, a sequence substantiallyidentical to the sequence used for primer design, described above, alsowas isolated. The nucleotide sequence of the gene is presented below.

FAO-18 (SEQ ID NO: 6)atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccgacaaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatcttgaccaatgttttgggatcaagggtcttggcaccagctttgaccaactcgttgactcctatcaaggacatgagcttggaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgctaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggggccggtgctggtgtcgtggcccacactttgaccaacgacggcttcaagagtttggttttggaaaagggcagatactttagcaactccgagttgaactttgatgacaaggacggggttcaagaattataccaaagtggaggtactttgaccaccgtcaaccagcagttgtttgttcttgctggttccacttttggtggtggtaccactgtcaattggtcggcctgtcttaaaacgccattcaaggtgcgtaaggaatggtatgatgagtttggcgttgactttgctgccgatgaagcctacgacaaagcacaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggcaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcatcacagatgcggtttctgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtttagagacgcagctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaagaagggcatcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggccccaaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattcaagaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagatcacttccacaactccatcatgactgctctttgttcagaagccgctgatttagacggcaagggtcatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggccatgttacttcttagtcgtgataccaccagtggttccgtttcgtcccatccaactaaacctgaagcattagttgtcgagtacgacgtgaacaagtttgacagaaactccatcttgcaggcattgttggtcactgctgacttgttgtacattcaaggtgccaagagaatccttagtccccaaccatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattccttttgacacctacggctcgccttatggttcggcgcatcaaatgtcttcttgtcgtatgtcaggtaagggtcctaaatacggtgctgttgataccgatggtagattgtttgaatgttcgaatgtttatgttgctgacgctagtcttttgccaactgctagcggtgctaatcctatggtcaccaccatgactcttgcaagacatgttgcgttaggtttggcagactccttgaagaccaaggccaagttgtag

Clustal nucleotide sequence alignments CLUSTAL 2.0.12 multiple sequencealignment FAO-13ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTA 60 FAO-20ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTA 60 FAO-17ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTA 60 FAO-18ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTA 60 FAO2aATGAATACCTTCTTGCCAGACGTGCTCGAATACAAACACGTCGACACCCTTTTGTTATTG 60 FAO2bATGAATACCTTCTTGCCAGACGTGCTCGAATACAAACACGTCGATACCCTTTTGTTATTA 60 ***  * *** ***** ***  * **** ************** ****** ******* FAO-13TGTGACGGGATCATCCACGAAACCACCGTCGACCAAATCAAAGACGTTATTGCTCCTGAC 120 FAO-20TGTGACGGGATCATCCACGAAACCACCGTCGACCAAATCAAAGACGTTATTGCTCCTGAC 120 FAO-17TGTGACGGGATCATCCACGAAACCACCGTGGACGAAATCAAAGACGTCATTGCCCCTGAC 120 FAO-18TGTGACGGGATCATCCACGAAACCACCGTGGACGAAATCAAAGACGTCATTGCCCCTGAC 120 FAO2aTGTGACGGGATCATCCACGAAACCACAGTCGATCAGATCAAGGACGCCATTGCTCCCGAC 120 FAO2bTGTGACGGGATCATCCACGAAACCACAGTCGACCAGATCAGGGACGCCATTGCTCCCGAC 120************************** ** **  * ****  ****  ***** ** *** FAO-13TTCCCTGCTGACAAGTACGAAGAGTACGTCAGGACATTCACCAAACCCTCCGAAACCCCA 180 FAO-20TTCCCTGCTGACAAGTACGAAGAGTACGTCAGGACATTCACCAAACCCTCCGAAACCCCA 180 FAO-17TTCCCCGCCGACAAATACGAGGAGTACGTCAGGACATTCACCAAACCCTCCGAAACCCCA 180 FAO-18TTCCCCGCCGACAAATACGAGGAGTACGTCAGGACATTCACCAAACCCTCCGAAACCCCA 180 FAO2aTTCCCTGAGGACCAGTACGAGGAGTATCTCAAGACCTTCACCAAGCCATCTGAGACCCCT 180 FAO2bTTCCCTGAAGACCAGTACGAGGAGTATCTCAAGACCTTCACCAAGCCATCTGAGACCCCT 180 ******  *** * ***** *****  *** *** ******** ** ** ** ***** FAO-13GGGTTCAGGGAAACCGTCTACAACACAGTCAACGCAAACACCACGGACGCAATCCACCAG 240 FAO-20GGGTTCAGGGAAACCGTCTACAACACAGTCAACGCAAACACCACGGACGCAATCCACCAG 240 FAO-17GGGTTCAGGGAAACCGTCTACAACACCGTCAACGCAAACACCATGGATGCAATCCACCAG 240 FAO-18GGGTTCAGGGAAACCGTCTACAACACCGTCAACGCAAACACCATGGATGCAATCCACCAG 240 FAO2aGGGTTCAGAGAAGCCGTCTACGACACGATCAACGCCACCCCAACCGATGCCGTGCACATG 240 FAO2bGGGTTCAGAGAAGCCGTCTACGACACGATCAACAGCACCCCAACCGAGGCTGTGCACATG 240******** *** ******** ****  *****   * * * *  ** **  * ***  * FAO-13TTCATTATCTTGACCAATGTTTTGGCATCCAGGGTCTTGGCTCCAGCTTTGACCAACTCG 300 FAO-20TTCATTATCTTGACCAATGTTTTGGCATCCAGGGTCTTGGCTCCAGCTTTGACCAACTCG 300 FAO-17TTCATTATCTTGACCAATGTTTTGGGATCAAGGGTCTTGGCACCAGCTTTGACCAACTCG 300 FAO-18TTCATTATCTTGACCAATGTTTTGGGATCAAGGGTCTTGGCACCAGCTTTGACCAACTCG 300 FAO2aTGTATTGTCTTGACCACCGCATTGGACTCCAGAATCTTGGCCCCCACGTTGACCAACTCG 300 FAO2bTGTATTGTATTGACCACCGCATTGGACTCGAGAATCTTGGCCCCCACGTTGACCAACTCG 300*  *** * *******  *  ****  ** **  ******* **  * ************ FAO-13TTGACGCCTATCAAGGACATGAGCTTGGAAGACCGTGAAAAATTGTTGGCCTCGTGGCGC 360 FAO-20TTGACGCCTATCAAGGACATGAGCTTGGAAGACCGTGAAAAATTGTTGGCCTCGTGGCGC 360 FAO-17TTGACTCCTATCAAGGACATGAGCTTGGAAGACCGTGAAAAGTTGTTAGCCTCGTGGCGT 360 FAO-18TTGACTCCTATCAAGGACATGAGCTTGGAAGACCGTGAAAAGTTGTTAGCCTCGTGGCGT 360 FAO2aTTGACGCCTATCAAGGATATGACCTTGAAGGAGCGTGAACAATTGTTGGCCTCTTGGCGT 360 FAO2bTTGACGCCTATCAAGGATATGACCTTGAAAGAGCGTGAACAATTGTTGGCTGCCTGGCGT 360 **************** **** **** * ** ****** * ***** **  * ***** FAO-13GACTCCCCAATCGCTGCCAAAAGGAAGTTGTTCAGGTTGGTTTCTACGCTTACCTTGGTC 420 FAO-20GACTCCCCAATCGCTGCCAAAAGGAAATTGTTCAGGTTGGTTTCCACGCTTACCTTGGTT 420 FAO-17GACTCCCCTATTGCTGCTAAAAGGAAGTTGTTCAGGTTGGTTTCTACGCTTACCTTGGTC 420 FAO-18GACTCCCCTATTGCTGCTAAAAGGAAGTTGTTCAGGTTGGTTTCTACGCTTACCTTGGTC 420 FAO2aGATTCCCCGATTGCGGCAAAGAGAAGATTGTTCAGATTGATTTCCTCGCTTACCTTGACG 420 FAO2bGATTCCCCGATCGCGGCCAAGAGAAGATTGTTCAGATTGATTTCCTCACTTACCTTGACG 420 ******* ** ** ** ** ** *  ******** *** ****  * ********* FAO-13ACGTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCATTATCCAGGAAGAGAA 480 FAO-20ACTTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCACTATCCAGGAAGAGAA 480 FAO-17ACGTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCATTATCCAGGAAGAGAA 480 FAO-18ACGTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCATTATCCAGGAAGAGAA 480 FAO2aACGTTTACGAGATTGGCCAGCGAATTGCACTTGAAAGCCATCCACTACCCTGGCAGAGAC 480 FAO2bACCTTTACGAGATTGGCCAGCGACTTGCACTTGAGAGCCATCCACTACCCTGGCAGAGAC 480 ** ***************  ** ***** **** ****** ** ** ** ** ***** FAO-13GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTTAAGTACCAGTTTTTGGAA 540 FAO-20GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTCAAGTACCAGTTTATGGAA 540 FAO-17GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTTAAGTACCAGTTTTTGGAA 540 FAO-18GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTTAAGTACCAGTTTTTGGAA 540 FAO2aTTGCGTGAAAAGGCGTATGAAACCCAGGTGGTTGACCCTTTCAGGTACCTGTTTATGGAG 540 FAO2bTTGCGTGAAAAGGCATATGAAACCCAGGTGGTTGACCCTTTCAGGTACCTGTTTATGGAA 540   *********** ************* * ********** * ***** **** **** FAO-13AAACCGAAGTTTTACGGCGCTGAGTTGTACTTGCCAGATATTGATGTGATCATTATTGGA 600 FAO-20AAGCCAAAGTTTGACGGCGCTGAGTTGTACTTGCCAGATATTGATGTTATCATTATTGGA 600 FAO-17AAACCGAAGTTTTACGGCGCTGAGTTGTACTTGCCAGATATTGATGTGATCATTATTGGA 600 FAO-18AAACCGAAGTTTTACGGCGCTGAGTTGTACTTGCCAGATATTGATGTGATCATTATTGGA 600 FAO2aAAACCAAAGTTTGACGGCGCCGAATTGTACTTGCCAGATATCGACGTCATCATCATTGGA 600 FAO2bAAACCAAAGTTTGACGGCACCGAGTTGTACTTGCCAGATATCGACGTCATCATCATTGGA 600 ** ******** ***** * ** ***************** ** ** ***** ****** FAO-13TCTGGTGCCGGTGCTGGTGTTGTGGCCCACACTTTGGCCAACGATGGCTTCAAGAGTTTG 660 FAO-20TCTGGTGCCGGTGCTGGTGTTGTGGCCCACACTTTGGCCAACGATGGCTTCAAGAGTTTG 660 FAO-17TCTGGTGCCGGTGCTGGTGTTGTGGCCCACACTTTGGCCAACGATGGCTTCAAGAGTTTG 660 FAO-18TCTGGGGCCGGTGCTGGTGTCGTGGCCCACACTTTGACCAACGACGGCTTCAAGAGTTTG 660 FAO2aTCAGGCGCCGGTGCTGGTGTCATGGCCCACACTCTCGCCAACGACGGGTTCAAGACCTTG 660 FAO2bTCCGGTGCCGGTGCTGGTGTCATGGCCCACACTTTAGCCAACGACGGGTACAAGACCTTG 660 ** ****************  *********** *  ******* ** * *****  *** FAO-13GTTTTGGAAAAGGGCAAATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGGC 720 FAO-20GTTTTGGAAAAGGGCAAATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGGC 720 FAO-17GTTTTGGAAAAGGGCAAATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGGC 720 FAO-18GTTTTGGAAAAGGGCAGATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGGG 720 FAO2aGTTTTGGAAAAGGGAAAGTATTTCAGCAACTCCGAGTTGAACTTTAATGACGCTGATGGC 720 FAO2bGTTTTGGAAAAGGGAAAGTATTTCAGCAACTCCGAGTTGAACTTTAATGATGCCGATGGT 720************** *  ** ** ********************* ****    ** ** FAO-13GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACTACAGTCAACCAACAGTTGTTTGTT 780 FAO-20GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACTACAGTCAACCAACAGTTGTTTGTT 780 FAO-17GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACTACAGTCAACCAACAGTTGTTTGTT 780 FAO-18GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACCACCGTCAACCAGCAGTTGTTTGTT 780 FAO2aGTGAAAGAGTTGTACCAAGGTAAAGGTGCTTTGGCCACCACCAATCAGCAGATGTTTATT 780 FAO2bATGAAAGAGTTGTACCAAGGTAAATGTGCGTTGACCACCACGAACCAGCAGATGTTTATT 780 *  **** ** ****** **  * ** * *** * **    ** ** *** ***** ** FAO-13CTTGCTGGTTCCACTTTTGGTGGCGGTACCACTGTCAATTGGTCAGCCTGTCTTAAGACG 840 FAO-20CTTGCTGGTTCCACTTTTGGTGGCGGTACCACTGTCAATTGGTCAGCCTGTCTTAAGACG 840 FAO-17CTTGCTGGTTCCACTTTTGGTGGCGGTACCACTGTCAATTGGTCAGCCTGTCTTAAGACG 840 FAO-18CTTGCTGGTTCCACTTTTGGTGGTGGTACCACTGTCAATTGGTCGGCCTGTCTTAAAACG 840 FAO2aCTTGCCGGTTCCACTTTGGGCGGTGGTACCACTGTCAACTGGTCTGCTTGCCTTAAAACA 840 FAO2bCTTGCCGGTTCCACTTTGGGCGGTGGTACCACTGTTAACTGGTCTGCTTGTCTTAAAACA 840 **************** ** ** *********** ** ***** ** ** ***** ** FAO-13CCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGTGTTGACTTTGCTGCTGATGAA 900 FAO-20CCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGTGTTGACTTTGCTGCTGATGAA 900 FAO-17CCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGTGTTGACTTTGCTGCTGATGAA 900 FAO-18CCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGCGTTGACTTTGCTGCCGATGAA 900 FAO2aCCATTTAAAGTGCGTAAGGAGTGGTACGACGAGTTTGGTCTTGAATTTGCTGCCGATGAA 900 FAO2bCCATTTAAAGTGCGTAAGGAGTGGTACGACGAGTTTGGTCTTGAATTTGCTGCCGACGAA 900 ******* *********** ***** ** ********  **** ******** ** *** FAO-13GCATACGATAAAGCGCAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCATC 960 FAO-20GCATACGATAAAGCGCAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCATC 960 FAO-17GCATACGATAAAGCGCAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCATC 960 FAO-18GCCTACGACAAAGCACAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCATC 960 FAO2aGCCTACGACAAAGCGCAGGATTATGTTTGGAAACAAATGGGTGCTTCAACAGATGGAATC 960 FAO2bGCCTACGACAAAGCACAAGACTATGTTTGGAAACAAATGGGCGCTTCTACCGAAGGAATC 960 ******* ***** ** ** ********* * ******** ***** ** ** ** *** FAO-13ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGTAAGAAATTAGGTTACAAGGCC 1020 FAO-20ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGTAAGAAATTAGGTTACAAGGCC 1020 FAO-17ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGTAAGAAATTAGGTTACAAGGCC 1020 FAO-18ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGCAAGAAATTAGGTTACAAGGCC 1020 FAO2aACTCACTCCTTGGCCAACGAAGTTGTGGTTGAAGGAGGTAAGAAGTTGGGCTACAAGAGC 1020 FAO2bACTCACTCTTTGGCGAACGCGGTTGTGGTTGAAGGAGGTAAGAAGTTGGGTTACAAGAGC 1020 ******* ***** ****   ** *  ******* ** ***** ** ** ******  * FAO-13AAGGTATTAGACCAAAACAGCGGTGGTCATCCTCAGCACAGATGCGGTTTCTGTTATTTG 1080 FAO-20AAGGTATTAGACCAAAACAGCGGTGGTCATCCTCAGCACAGATGCGGTTTCTGTTATTTG 1080 FAO-17AAGGTATTAGACCAAAACAGCGGTGGTCATCCTCAGCACAGATGCGGTTTCTGTTATTTG 1080 FAO-18AAGGTATTAGACCAAAACAGCGGTGGTCATCCTCATCACAGATGCGGTTTCTGTTATTTG 1080 FAO2aAAGGAAATTGAGCAGAACAACGGTGGCCACCCTGACCACCCATGTGGTTTCTGTTACTTG 1080 FAO2bAAGGAAATCGAGCAGAACAATGGTGGCCATCCTGACCACCCCTGTGGTTTCTGTTACTTG 1080**** * * ** ** ****  ***** ** *** * ***   ** *********** *** FAO-13GGCTGTAAGCACGGTATCAAGCAGGGTTCTGTTAATAACTGGTTTAGAGACGCAGCTGCC 1140 FAO-20GGCTGTAAGCACGGTATCAAGCAGGGTTCTGTTAATAACTGGTTTAGAGACGCAGCTGCC 1140 FAO-17GGTTGTAAGCACGGTATCAAGCAGGGCTCTGTTAATAACTGGTTTAGAGACGCAGCTGCC 1140 FAO-18GGTTGTAAGCACGGTATCAAGCAGGGCTCTGTTAATAACTGGTTTAGAGACGCAGCTGCC 1140 FAO2aGGCTGTAAGTACGGTATTAAACAGGGTTCTGTGAATAACTGGTTTAGAGACGCAGCTGCC 1140 FAO2bGGCTGTAAGTACGGTATTAAGCAGGGTTCTGTGAATAACTGGTTTAGAGACGCAGCTGCC 1140 ******** ******* ** ***** ***** *************************** FAO-13CACGGTTCCCAGTTCATGCAACAGGTTAGAGTTTTGCAAATACTTAACAAGAAGGGGATC 1200 FAO-20CACGGTTCCCAGTTCATGCAACAGGTTAGAGTTTTGCAAATACTTAACAAGAAGGGGATC 1200 FAO-17CACGGTTCTCAGTTCATGCAACAGGTTAGAGTTTTGCAAATCCTTAACAAGAAGGGCATC 1200 FAO-18CACGGTTCTCAGTTCATGCAACAGGTTAGAGTTTTGCAAATCCTTAACAAGAAGGGCATC 1200 FAO2aCACGGGTCCAAGTTCATGCAACAAGTCAGAGTTGTGCAAATCCTCAACAAGAATGGCGTC 1200 FAO2bCACGGGTCCAAGTTCATGCAACAAGTCAGAGTTGTGCAAATCCTCCACAATAAAGGCGTC 1200 *******  ************* ** ****** ******* **  **** ** **  ** FAO-13GCTTACGGTATCTTGTGTGAGGATGTTGTAACCGGCGCCAAGTTCACCATTACTGGCCCC 1260 FAO-20GCTTACGGTATCTTGTGTGAGGATGTTGTAACCGGCGCCAAGTTCACCATTACTGGCCCC 1260 FAO-17GCTTATGGTATCTTGTGTGAGGATGTTGTAACCGGTGCCAAGTTCACCATTACTGGCCCC 1260 FAO-18GCTTATGGTATCTTGTGTGAGGATGTTGTAACCGGTGCCAAGTTCACCATTACTGGCCCC 1260 FAO2aGCTTATGGTATCTTGTGTGAGGATGTCGAAACCGGAGTCAGGTTCACTATTAGTGGCCCC 1260 FAO2bGCTTATGGCATCTTGTGTGAGGATGTCGAGACCGGAGTCAAATTCACTATCAGTGGCCCC 1260 ******* ***************** *  ***** * **  ***** ** * ******* FAO-13AAAAAGTTTGTTGTTGCTGCCGGTGCTTTGAACACTCCATCTGTGTTGGTCAACTCCGGC 1320 FAO-20AAAAAGTTTGTTGTTGCTGCCGGTGCTTTGAACACTCCATCTGTGTTGGTCAACTCCGGC 1320 FAO-17AAAAAGTTTGTTGTTGCCGCCGGCGCCTTAAACACTCCATCTGTGTTGGTCAACTCCGGA 1320 FAO-18AAAAAGTTTGTTGTTGCCGCCGGCGCCTTAAACACTCCATCTGTGTTGGTCAACTCCGGA 1320 FAO2aAAAAAGTTTGTTGTTTCTGCTGGTTCTTTGAACACGCCAACTGTGTTGACCAACTCCGGA 1320 FAO2bAAAAAGTTTGTTGTTTCTGCAGGTTCTTTGAACACGCCAACGGTGTTGACCAACTCCGGA 1320*************** * ** **  * ** ***** *** * ******  ********* FAO-13TTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCACCCAGTTTCTGTCGTGTTTGGT 1380 FAO-20TTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCACCCAGTTTCTGTCGTGTTTGGT 1380 FAO-17TTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCATCCAGTTTCTGTCGTGTTTGGT 1380 FAO-18TTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCATCCAGTTTCTGTCGTGTTTGGT 1380 FAO2aTTCAAGAACAAGCACATTGGTAAGAACTTGACGTTGCACCCAGTTTCCACCGTGTTTGGT 1380 FAO2bTTCAAGAACAAACACATCGGTAAGAACTTGACGTTGCACCCAGTTTCGACCGTGTTTGGT 1380***********  **** *********** ** ***** ********   ********** FAO-13GATTTTGGCAAAGACGTTCAAGCAGACCACTTCCACAACTCCATCATGACTGCCCTTTGT 1440 FAO-20GATTTTGGCAAAGACGTTCAAGCAGACCACTTCCACAACTCCATCATGACTGCCCTTTGT 1440 FAO-17GATTTTGGCAAAGACGTTCAAGCAGACCACTTCCACAACTCCATCATGACTGCCCTTTGT 1440 FAO-18GATTTTGGCAAAGACGTTCAAGCAGATCACTTCCACAACTCCATCATGACTGCTCTTTGT 1440 FAO2aGACTTTGGCAGAGACGTGCAAGCCGACCATTTCCACAAATCTATTATGACTTCGCTTTGT 1440 FAO2bGACTTTGGCAGAGACGTGCAAGCCGACCATTTCCACAAATCTATTATGACTTCGCTCTGT 1440 ********* ****** ***** ** ** ******** ** ** ****** * ** *** FAO-13TCAGAAGCCGCTGATTTAGACGGCAAGGGCCATGGATGCAGAATTGAAACCATCTTGAAC 1500 FAO-20TCAGAAGCCGCTGATTTAGACGGCAAGGGCCATGGATGCAGAATTGAAACCATCTTGAAC 1500 FAO-17TCAGAAGCCGCTGATTTAGACGGCAAGGGCCATGGATGCAGAATTGAAACCATCTTGAAC 1500 FAO-18TCAGAAGCCGCTGATTTAGACGGCAAGGGTCATGGATGCAGAATTGAAACCATCTTGAAC 1500 FAO2aTACGAGGTTGCTGACTTGGACGGCAAGGGCCACGGATGCAGAATCGAAACCATCTTGAAC 1500 FAO2bTACGAAGTCGCTGACTTGGACGGCAAGGGCCACGGATGCAGAATCGAGACCATCTTGAAC 1500 *  ***  ***** ** *********** ** *********** ** ************ FAO-13GCTCCATTCATCCAGGCTTCATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGAC 1560 FAO-20GCTCCATTCATCCAGGCTTCATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGAC 1560 FAO-17GCTCCATTCATCCAGGCTTCATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGAC 1560 FAO-18GCTCCATTCATCCAGGCTTCATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGAC 1560 FAO2aGCTCCATTCATCCAAGCTTCTTTGTTGCCATGGAGAGGAAGTGACGAGGTCAGAAGAGAC 1560 FAO2bGCTCCATTCATCCAAGCTTCTTTGTTGCCATGGAGAGGAAGCGACGAGGTCAGAAGAGAC 1560************** ***** ** ** *********** **  ******  *** ***** FAO-13TTGTTGCGTTACAACAACATGGTGGCGATGTTGCTCCTTAGTCGTGACACCACCAGTGGT 1620 FAO-20TTGTTGCGTTACAACAACATGGTGGCGATGTTGCTCCTTAGTCGTGACACCACCAGTGGT 1620 FAO-17TTGTTGCGTTACAACAACATGGTGGCGATGTTGCTCCTTAGTCGTGACACCACCAGTGGT 1620 FAO-18TTGTTGCGTTACAACAACATGGTGGCCATGTTACTTCTTAGTCGTGATACCACCAGTGGT 1620 FAO2aTTGTTGCGTTACAACAACATGGTGGCCATGTTGCTTATCACGCGTGATACCACCAGTGGT 1620 FAO2bTTGTTGCGTTACAACAACATGGTGGCCATGTTGCTTATCACCCGTGACACCACCAGTGGT 1620************************** ***** **  * *  ***** ************ FAO-13TCCGTTTCTGCTCATCCAACCAAACCTGAAGCTTTGGTTGTCGAGTACGACGTGAACAAG 1680 FAO-20TCCGTTTCTGCTCATCCAACCAAACCTGAAGCTTTGGTTGTCGAGTACGACGTGAACAAG 1680 FAO-17TCCGTTTCTGCTCATCCAACCAAACCTGAAGCTTTGGTTGTCGAGTACGACGTGAACAAG 1680 FAO-18TCCGTTTCGTCCCATCCAACTAAACCTGAAGCATTAGTTGTCGAGTACGACGTGAACAAG 1680 FAO2aTCAGTTTCTGCTGACCCAAAGAAGCCCGACGCTTTGATTGTCGACTATGAGATTAACAAG 1680 FAO2bTCAGTTTCTGCTGACCCAAAGAAGCCCGACGCTTTGATTGTCGACTATGACATCAACAAG 1680 *******  *  * ****  ** ** ** ** **  ******* ** **  * ****** FAO-13TTTGACAGAAACTCGATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTATATCCAA 1740 FAO-20TTTGACAGAAACTCGATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTATATCCAA 1740 FAO-17TTTGACAGAAACTCGATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTATATCCAA 1740 FAO-18TTTGACAGAAACTCCATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTACATTCAA 1740 FAO2aTTTGACAAGAATGCCATCTTGCAAGCTTTCTTGATCACTTCCGACATGTTGTACATTGAA 1740 FAO2bTTTGACAAGAATGCCATCTTGCAAGCTTTCTTGATCACCTCCGACATGTTGTACATCGAA 1740*******  **  * ******** ** ** *** ****  * *** ******* **  ** FAO-13GGTGCCAAGAGAATCCTTAGTCCACAGGCATGGGTGCCAATTTTTGAATCCGACAAGCCA 1800 FAO-20GGTGCCAAGAGAATCCTTAGTCCACAGGCATGGGTGCCAATTTTTGAATCCGACAAGCCA 1800 FAO-17GGTGCCAAGAGAATCCTTAGTCCACAGGCATGGGTGCCAATTTTTGAATCCGACAAGCCA 1800 FAO-18GGTGCCAAGAGAATCCTTAGTCCCCAACCATGGGTGCCAATTTTTGAATCCGACAAGCCA 1800 FAO2aGGTGCCAAGAGAATCCTCAGTCCACAGCCATGGGTGCCAATCTTTGAGTCGAACAAGCCA 1800 FAO2bGGTGCCAAGAGAATCCTCAGTCCACAGGCATGGGTGCCAATCTTTGAGTCGAACAAGCCA 1800***************** ***** **  ************* ***** **  ******** FAO-13AAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAATGGAGAGCCAAGGTTGCCAAG 1860 FAO-20AAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAATGGAGAGCCAAGGTTGCCAAG 1860 FAO-17AAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAATGGAGAGCCAAGGTTGCCAAG 1860 FAO-18AAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAATGGAGAGCCAAGGTTGCCAAG 1860 FAO2aAAGGAGCAAAGAACGATCAAGGACAAGGACTATGTTGAGTGGAGAGCCAAGGCTGCTAAG 1860 FAO2bAAGGAGCAAAGAACAATCAAGGACAAGGACTATGTCGAATGGAGAGCCAAGGCTGCCAAG 1860*****  * *** * ********* ********** ** ************* *** *** FAO-13ATTCCTTTCGACACCTACGGCTCACCTTATGGTTCGGCACATCAAATGTCTTCTTGCCGT 1920 FAO-20ATTCCTTTCGACACCTACGGCTCACCTTATGGTTCGGCACATCAAATGTCTTCTTGCCGT 1920 FAO-17ATTCCTTTCGACACCTACGGCTCACCTTATGGTTCGGCACATCAAATGTCTTCTTGCCGT 1920 FAO-18ATTCCTTTTGACACCTACGGCTCGCCTTATGGTTCGGCGCATCAAATGTCTTCTTGTCGT 1920 FAO2aATACCTTTCGACACCTACGGTTCTGCATATGGGTCCGCACATCAAATGTCCACCTGTCGT 1920 FAO2bATACCTTTCGACACCTACGGTTCTGCCTATGGGTCCGCACATCAAATGTCCACCTGTCGT 1920 ******* *********** **  * ***** ** ** ***********  * ** *** FAO-13ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGACACCGATGGTAGATTGTTTGAATGT 1980 FAO-20ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGACACCGATGGTAGATTGTTTGAATGT 1980 FAO-17ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGACACCGATGGTAGATTGTTTGAATGT 1980 FAO-18ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGATACCGATGGTAGATTGTTTGAATGT 1980 FAO2aATGTCCGGAAAGGGTCCTAAATACGGTGCTGTTGATACTGATGGTAGATTGTTTGAATGT 1980 FAO2bATGTCCGGAAAGGGTCCTAAATACGGCGCCGTTGATACCGATGGTAGATTGTTTGAATGT 1980 ******* ***************** ** ***** ** ********************* FAO-13TCGAATGTTTATGTTGCCGATGCAAGTCTTTTGCCAACTGCAAGCGGTGCCAACCCTATG 2040 FAO-20TCGAATGTTTATGTTGCCGATGCAAGTCTTTTGCCAACTGCAAGCGGTGCCAACCCTATG 2040 FAO-17TCGAATGTTTATGTTGCCGATGCAAGTCTTTTGCCAACTGCAAGCGGTGCCAACCCTATG 2040 FAO-18TCGAATGTTTATGTTGCTGACGCTAGTCTTTTGCCAACTGCTAGCGGTGCTAATCCTATG 2040 FAO2aTCGAATGTCTATGTTGCTGATGCTAGTGTTTTGCCTACTGCCAGCGGTGCCAACCCAATG 2040 FAO2bTCGAATGTCTATGTTGCTGATGCTAGTGTTTTGCCTACTGCCAGCGGTGCCAACCCAATG 2040******** ******** ** ** *** ******* ***** ******** ** ** *** FAO-13GTCACCACCATGACTCTTGCCAGACATGTTGCGTTAGGTTTGGCAGACTCCTTGAAGACC 2100 FAO-20GTCACCACCATGACTCTTGCCAGACATGTTGCGTTAGGTTTGGCAGACTCCTTGAAGACC 2100 FAO-17GTCACCACCATGACTCTTGCAAGACATGTTGCGTTAGGTTTGGCAGACTCCTTGAAGACC 2100 FAO-18GTCACCACCATGACTCTTGCAAGACATGTTGCGTTAGGTTTGGCAGACTCCTTGAAGACC 2100 FAO2aATATCCACCATGACCTTTGCTAGACAGATTGCGTTAGGTTTGGCTGACTCCTTGAAGACC 2100 FAO2bATCTCCACCATGACGTTTGCTAGACAGATTGCGTTAGGTTTGGCTGACTCTTTGAAGACC 2100 *  **********  **** *****  **************** ***** ********* FAO-13AAAGCCAAGTTGTAG 2115 FAO-20 AAAGCCAAGTTGTAG 2115 FAO-17 AAGGCCAAGTTGTAG2115 FAO-18 AAGGCCAAGTTGTAG 2115 FAO2a AAACCCAAGTTGTAG 2115 FAO2bAAACCCAAGTTGTAG 2115 **  ***********

Amino acid sequences FAO-1 SEQ ID NO: 7MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLTNDGFKSLVLEKGRYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL FAO-13SEQ ID NO: 8MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL FAO-20SEQ ID NO: 9MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFMEKPKFDGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL FAO-17SEQ ID NO: 10MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKl FAO-2aSEQ ID NO: 11MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIKDAIAPDFPEDQYEEYLKTFTKPSETPGFREAVYDTINATPTDAVHMCIVLTTALDSRILAPTLTNSLTPIKDMTLKEREQLLASWRDSPIAAKRRLFRLISSLTLTTFTRLASELHLKAIHYPGRDLREKAYETQVVDPFRYSFMEKPKFDGAELYLPDIDVIIIGSGAGAGVMAHTLANDGFKTLVLEKGKYFSNSELNFNDADGVKELYQGKGALATTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTDGITHSLANEVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILNKNGVAYGILCEDVETGVRFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYEINKFDKNAILQAFLITSDMLYIEGAKRILSPQPWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMSTCRMSGKGPKYGAVDTDGRLFECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL FAO-2bSEQ ID NO: 12MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIRDAIAPDFPEDQYEEYLKTFTKPSETPGFREAVYDTINSTPTEAVHMCIVLTTALDSRILAPTLTNSLTPIKDMTLKEREQLLAAWRDSPIAAKRRLFRLISSLTLTTFTRLASDLHLRAIHYPGRDLREKAYETQVVDPFRYSFMEKPKFDGTELYLPDIDVIIIGSGAGAGVMAHTLANDGYKTLVLEKGKYFSNSELNFNDADGMKELYQGKCALTTTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTEGITHSLANAVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILHNKGVAYGILCEDVETGVKFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYDINKFDKNAILQAFLITSDMLYIEGAKRILSPQAWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMSTCRMSGKGPKYGAVDTDGRLFECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL

Clustal amino acid sequence alignments FAO-13MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETP 60 FAO-20MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETP 60 FAO-1MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETP 60*******************************:**************************** FAO-13GFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWR 120 FAO-20GFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWR 120 FAO-1GFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWR 120************** *************.******************************* FAO-13DSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLE 180 FAO-20DSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFME 180 FAO-1DSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLE 180**********************************************************:* FAO-13KPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDG 240 FAO-20KPKFDGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDG 240 FAO-1KPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLTNDGFKSLVLEKGRYFSNSELNFDDKDG 240 *******************************:************:************** FAO-13VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADE 300 FAO-20VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADE 300 FAO-1VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADE 300************************************************************ FAO-13AYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYL 360 FAO-20AYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYL 360 FAO-1AYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYL 360***************************************************:******** FAO-13GCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGP 420 FAO-20GCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGP 420 FAO-1GCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGP 420************************************************************ FAO-13KKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALC 480 FAO-20KKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALC 480 FAO-1KKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALC 480************************************************************ FAO-13SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG 540 FAO-20SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG 540 FAO-1SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG 540************************************************************ FAO-13SVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKP 600 FAO-20SVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKP 600 FAO-1SVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKP 600***:*********************************************.********** FAO-13KDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFEC 660 FAO-20KDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFEC 660 FAO-1KDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFEC 660************************************************************ FAO-13SNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL 704 FAO-20SNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL 704 FAO-1SNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL 704********************************************

Example 31 Examples of Embodiments

Provided hereafter are non-limiting examples of certain embodiments.

A1. An engineered microorganism capable of producing adipic acid, whichmicroorganism comprises one or more altered activities selected from thegroup consisting of 6-oxohexanoic acid dehydrogenase activity, omega oxofatty acid dehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenaseactivity, omega hydroxyl fatty acid dehydrogenase activity, hexanoatesynthase activity, monooxygenase activity, monooxygenase reductaseactivity, fatty alcohol oxidase activity, acyl-CoA ligase activity,acyl-CoA oxidase activity, enoyl-CoA hydratase activity,3-L-hydroxyacyl-CoA dehydrogenase activity and acetyl-CoAC-acyltransferase activity.

A1.1. The engineered microorganism of embodiment A1, which comprises agenetic modification that adds or increases the 6-oxohexanoic aciddehydrogenase activity, omega oxo fatty acid dehydrogenase activity,6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity, hexanoate synthase activity, monooxygenaseactivity, monooxygenase reductase activity, fatty alcohol oxidaseactivity, acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoAhydratase activity, 3-L-hydroxyacyl-CoA dehydrogenase activity and/oracetyl-CoA C-acyltransferase activity.

A1.2. The engineered microorganism of embodiment A1, which comprises agenetic modification that reduces the acyl-CoA oxidase activity.

A1.3. The engineered microorganism of embodiment A1.1, wherein thegenetic modification comprises increased copies of a polynucleotide thatencodes a polypeptide having 6-oxohexanoic acid dehydrogenase activity,omega oxo fatty acid dehydrogenase activity, 6-hydroxyhexanoic aciddehydrogenase activity, omega hydroxyl fatty acid dehydrogenaseactivity, hexanoate synthase activity, monooxygenase activity,monooxygenase reductase activity, fatty alcohol oxidase activity,acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoA hydrataseactivity, 3-L-hydroxyacyl-CoA dehydrogenase activity or acetyl-CoAC-acyltransferase activity.

A1.4. The engineered microorganism of embodiment A1.1, wherein thegenetic modification comprises insertion of a heterologous promoterand/or 5′ UTR, into genomic DNA of the microorganism in functionalconnection with a polynucleotide that encodes a polypeptide having6-oxohexanoic acid dehydrogenase activity, 6-hydroxyhexanoic aciddehydrogenase activity, hexanoate synthase activity, monooxygenaseactivity, monooxygenase reductase activity, fatty alcohol oxidaseactivity, acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoAhydratase activity, 3-L-hydroxyacyl-CoA dehydrogenase activity oracetyl-CoA C-acyltransferase activity.

A2. The engineered microorganism of any one of embodiments A1 to A1.4,which comprises an altered thioesterase activity.

A2.1. The engineered microorganism of embodiment A2, which comprises agenetic alteration that adds or increases a thioesterase activity.

A2.2. The engineered microorganism of embodiment A2.1, which comprises aheterologous polynucleotide encoding a polypeptide having thioesteraseactivity.

A3. The engineered microorganism of any one of embodiments A1 to A2.2,which comprises a heterologous polynucleotide encoding a polypeptidehaving 6-oxohexanoic acid dehydrogenase activity.

A3.1 The engineered microorganism of any one of embodiments A1 to A3,which comprises a heterologous polynucleotide encoding a polypeptidehaving omega oxo fatty acid dehydrogenase activity.

A4. The engineered microorganism of embodiment A3 and A3.1, wherein theheterologous polynucleotide is from a bacterium.

A5. The engineered microorganism of embodiment A4, wherein the bacteriumis an Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium.

A6. The engineered microorganism of embodiment A1 or A2, which comprisesa heterologous polynucleotide encoding a polypeptide having6-hydroxyhexanoic acid dehydrogenase activity.

A6.1 The engineered microorganism of embodiment A1 or A2, whichcomprises a heterologous polynucleotide encoding a polypeptide havingomega hydroxyl fatty acid dehydrogenase activity

A7. The engineered microorganism of embodiment A6 or A6.1, wherein theheterologous polynucleotide is from a bacterium.

A8. The engineered microorganism of embodiment A7, wherein the bacteriumis an Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium.

A9. The engineered microorganism of embodiment A1 or A2, which comprisesa heterologous polynucleotide encoding a polypeptide having hexanoatesynthase subunit A activity.

A10. The engineered microorganism of embodiment A1 or A2, whichcomprises a heterologous polynucleotide encoding a polypeptide havinghexanoate synthase subunit B activity.

A11. The engineered microorganism of embodiment A9 or A10, wherein theheterologous polynucleotide independently is selected from a bacterium.

A12. The engineered microorganism of embodiment A11, wherein thebacterium is a Bacillus bacterium.

A13. The engineered microorganism of embodiment A12, wherein theBacillus bacterium is B. megaterium.

A14. The engineered microorganism of embodiment A1 or A2, whichcomprises a heterologous polynucleotide encoding a polypeptide havingmonooxygenase activity.

A15. The engineered microorganism of embodiment A14, wherein theheterologous polynucleotide is from a fungus.

A16. The engineered microorganism of embodiment A15, wherein the fungusis an Aspergillus fungus.

A17. The engineered microorganism of embodiment A16, wherein theAspergillus fungus is A. parasiticus.

A18. The engineered microorganism of embodiment A1 or A2, whichcomprises a genetic modification that results in primary hexanoate usageby monooxygenase activity.

A19. The engineered microorganism of embodiment A18, wherein the geneticmodification reduces a polyketide synthase activity.

A20. The engineered microorganism of any one of embodiments A1-A19,which is a eukaryote.

A21. The engineered microorganism of embodiment A20, which is a yeast.

A22. The engineered microorganism of embodiment A21, wherein the yeastis a Candida yeast.

A23. The engineered microorganism of embodiment A22, wherein the Candidayeast is a C. tropicalis strain.

A24. The engineered microorganism of embodiment A20, which is a fungus.

A25. The engineered microorganism of embodiment A24, wherein the fungusis a Yarrowia fungus.

A26. The engineered microorganism of embodiment A25, wherein theYarrowia fungus is Y. lipolytica.

A27. The engineered microorganism of embodiment A24, wherein the fungusis an Aspergillus fungus.

A28. The engineered microorganism of embodiment A27, wherein theAspergillus fungus is a A. parasiticus strain or a A. nidulans strain.

A29. The engineered microorganism of any one of embodiments A1-A28,which comprises a genetic modification that reduces 6-hydroxyhexanoicacid conversion.

A30. The engineered microorganism of embodiment A29, wherein the geneticmodification reduces 6-hydroxyhexanoic acid dehydrogenase activity.

A31. The engineered microorganism of any one of embodiments A1-A30,which comprises a genetic modification that reduces beta-oxidationactivity.

A32. The engineered microorganism of embodiment A31, wherein the geneticmodification renders beta-oxidation activity undetectable.

A33. The engineered microorganism of embodiment A31, wherein the geneticmodification partially reduces beta-oxidation activity.

A34. The engineered microorganism of any one of embodiments A31 to A33,wherein the genetic modification comprises disrupting a polynucleotidethat encodes a polypeptide having an acyl-CoA oxidase activity.

A35. The engineered microorganism of any one of embodiments A31 to A33,wherein the genetic modification comprises disrupting a promoter infunctional connection with a polynucleotide that encodes a polypeptidehaving the acyl-CoA oxidase activity.

A36. The engineered microorganism of embodiment A34 or A35, wherein thepolypeptide having the acyl-CoA oxidase activity is a POX polypeptide.

A37. The engineered microorganism of embodiment A36, wherein the POXpolypeptide is a POX4 polypeptide, POX5 polypeptide or POX4 polypeptideand POX5 polypeptide.

A38. The engineered microorganism of any one of embodiments A1 to A37,which is in contact with a feedstock.

A39. The engineered microorganism of embodiment A38, wherein thefeedstock comprises a saccharide.

A40. The engineered microorganism of embodiment A39, wherein thesaccharide is a monosaccharide, polysaccharide or a mixture or amonosaccharide and polysaccharide.

A41. The engineered microorganism of embodiment A38, wherein thefeedstock comprises a paraffin.

A42. The engineered microorganism of embodiment A41, wherein theparaffin is a saturated paraffin, unsaturated paraffin, substitutedparaffin, branched paraffin, linear paraffin or combination thereof.

A43. The engineered microorganism of embodiment A41 or A42, wherein theparaffin includes about 1 to about 60 carbon atoms.

A44. The engineered microorganism of embodiments A41 to A43, wherein theparaffin is in a mixture of paraffins.

A45. The engineered microorganism of embodiment A44, wherein theparaffins in the mixture of paraffins have a mean number of carbon atomsof about 8 to about 18 carbon atoms.

A46. The engineered microorganism of embodiment A45, wherein the meannumber of carbon atoms is about 10 to about 16 carbon atoms.

A46.1. The engineered microorganism of embodiment A46, wherein the meannumber of carbon atoms is about 12 atoms.

A47. The engineered microorganism of any one of embodiments A41 toA46.1, wherein the paraffin is in a wax.

A48. The engineered microorganism of any one of embodiments A41 toA46.1, wherein the paraffin is in an oil.

A49. The engineered microorganism of any one of embodiments A41 to A48,wherein the paraffin is from a petroleum product.

A50. The engineered microorganism of embodiment A49, wherein thepetroleum product is a petroleum distillate.

A51. The engineered microorganism of any one of embodiments A41 to A48,wherein the paraffin is from a plant or plant product.

B1. An engineered microorganism that produces adipic acid, whichmicroorganism comprises an altered monooxygenase activity.

B1.1. The engineered microorganism of embodiment B1, which comprises agenetic modification that alters the monooxygenase activity.

B1.2. The engineered microorganism of embodiment B1 or B1.1, whichcomprises a genetic modification that alters a monooxygenase activityselected from the group consisting of.

B2. The engineered microorganism of embodiment B1.1, which comprises aheterologous polynucleotide encoding a polypeptide having monooxygenaseactivity.

B3. The engineered microorganism of embodiment B2, wherein theheterologous polynucleotide is from a fungus.

B4. The engineered microorganism of embodiment B3, wherein the fungus isan Aspergillus fungus.

B5. The engineered microorganism of embodiment B4, wherein theAspergillus fungus is A. parasiticus.

B6. The engineered microorganism of any one of embodiments B1-B5, whichcomprises a genetic modification that results in substantial hexanoateusage by the monooxygenase activity.

B7. The engineered microorganism of embodiment B6, wherein the geneticmodification reduces a polyketide synthase activity.

B8. The engineered microorganism of any one of embodiments B1-B5, whichcomprises an altered hexanoate synthase activity.

B9. The engineered microorganism of embodiment B8, wherein the alteredhexanoate synthase activity is an altered hexanoate synthase subunit Aactivity, altered hexanoate synthase subunit B activity, or alteredhexanoate synthase subunit A activity and altered hexanoate synthasesubunit B activity.

B9.1. The engineered microorganism of embodiment B9, which comprises agenetic alteration that adds or increases hexanoate synthase activity.

B10. The engineered microorganism of any one of embodiments B8, B9 orB9.1, which comprises a heterologous polynucleotide encoding apolypeptide having hexanoate synthase activity.

B11. The engineered microorganism of embodiment B10, wherein theheterologous polynucleotide is from a fungus.

B12. The engineered microorganism of embodiment B11, wherein the fungusis an Aspergillus fungus.

B13. The engineered microorganism of embodiment B11, wherein theAspergillus fungus is A. parasiticus.

B14. The engineered microorganism of any one of embodiments B1-B13,which comprises an altered thioesterase activity.

B14.1. The engineered microorganism of embodiment B14, which comprises agenetic modification that adds or increases the thioesterase activity.

B14.2. The engineered microorganism of embodiment B14 or B14.1, whichcomprises a heterologous polynucleotide encoding a polypeptide havingthioesterase activity.

B15. The engineered microorganism of any one of embodiments B1-B14.2,which comprises an altered 6-oxohexanoic acid dehydrogenase activity.

B15.1. The engineered microorganism of embodiment B15, which comprises agenetic modification that adds or increases the 6-oxohexanoic aciddehydrogenase activity.

B15.2 The engineered microorganism of any one of embodiments B1 toB15.1, which comprises an altered omega oxo fatty acid dehydrogenaseactivity.

B15.3 The engineered microorganism of embodiment B15.2, which comprisesa genetic modification that adds or increases the omega oxo fatty aciddehydrogenase activity

B16. The engineered microorganism of any one of embodiments B15 toB15.3, which comprises a heterologous polynucleotide encoding apolypeptide having 6-oxohexanoic acid dehydrogenase activity.

B16.1 The engineered microorganism of any one of embodiments B15 to B16,which comprises a heterologous polynucleotide encoding a polypeptidehaving omega oxo fatty acid dehydrogenase activity.

B17. The engineered microorganism of embodiment B16 or B16.1, whereinthe heterologous polynucleotide is from a bacterium.

B18. The engineered microorganism of embodiment B17, wherein thebacterium is a Acinetobacter, Nocardia, Pseudomonas or Xanthobacterbacterium.

B19. The engineered microorganism of any one of embodiments B1-B18,which comprises an altered 6-hydroxyhexanoic acid dehydrogenaseactivity.

B19.1. The engineered microorganism of embodiment B19, which comprises agenetic modification that adds or increases the 6-hydroxyhexanoic aciddehydrogenase activity.

B19.2 The engineered microorganism of any one of embodiments B1-B19.1,which comprises an altered omega hydroxyl fatty acid dehydrogenaseactivity.

B19.3 The engineered microorganism of embodiment B19.2, which comprisesa genetic modification that adds or increases the omega hydroxyl fattyacid dehydrogenase activity.

B20. The engineered microorganism of any one of embodiments B19 toB19.3, which comprises a heterologous polynucleotide encoding apolypeptide having 6-hydroxyhexanoic acid dehydrogenase activity.

B20.1 The engineered microorganism of any one of embodiments B19 to B20,which comprises a heterologous polynucleotide encoding a polypeptidehaving omega hydroxyl fatty acid dehydrogenase activity.

B21. The engineered microorganism of embodiment B20 or B20.1, whereinthe heterologous polynucleotide is from a bacterium.

B22. The engineered microorganism of embodiment B21, wherein thebacterium is a Acinetobacter, Nocardia, Pseudomonas or Xanthobacterbacterium.

B23. The engineered microorganism of any one of embodiments B1-B22,which is a eukaryote.

B24. The engineered microorganism of embodiment B23, which is a yeast.

B25. The engineered microorganism of embodiment B24, wherein the yeastis a Candida yeast.

B26. The engineered microorganism of embodiment B25, wherein the Candidayeast is C. tropicalis.

B27. The engineered microorganism of embodiment B23, which is a fungus.

B28. The engineered microorganism of embodiment B27, wherein the fungusis a Yarrowia fungus.

B29. The engineered microorganism of embodiment B28, wherein theYarrowia fungus is Y. lipolytica.

B30. The engineered microorganism of embodiment B27, wherein the fungusis Aspergillus.

B31. The engineered microorganism of embodiment B30, wherein theAspergillus fungus is A. parasiticus or A. nidulans.

B32. The engineered microorganism of any one of embodiments B1-B31,which comprises a genetic modification that reduces 6-hydroxyhexanoicacid conversion.

B33. The engineered microorganism of embodiment B32, wherein the geneticmodification reduces 6-hydroxyhexanoic acid dehydrogenase activity.

B34. The engineered microorganism of any one of embodiments B1-B33,which comprises a genetic modification that reduces beta-oxidationactivity.

B35. The engineered microorganism of embodiment B34, wherein the geneticmodification renders beta-oxidation activity undetectable.

C1. A method for manufacturing adipic acid, which comprises culturing anengineered microorganism of any one of embodiments A1-B35 under cultureconditions in which the cultured microorganism produces adipic acid.

C1.1. The method of embodiment C1, wherein the host microorganism fromwhich the engineered microorganism is produced does not produce adetectable amount of adipic acid.

C2. The method of embodiment Cl of C1.1, wherein the culture conditionscomprise fermentation conditions.

C3. The method of any one of embodiments C1-C2, wherein the cultureconditions comprise introduction of biomass.

C4. The method of C1 or C2, wherein the culture conditions compriseintroduction of glucose.

C5. The method of C1 or C2, wherein the culture conditions compriseintroduction of hexane.

C6. The method of any one of embodiments C1-C5, wherein the adipic acidis produced with a yield of greater than about 0.3 grams per gram ofglucose added.

C7. The method of any one of embodiments C1-C6, which comprisespurifying the adipic acid from the cultured microorganisms.

C8. The method of embodiment C7, which comprises modifying the adipicacid, thereby producing modified adipic acid.

C9. The method of any one of embodiments C1-C8, which comprises placingthe cultured microorganisms, the adipic acid or the modified adipic acidin a container.

C10. The method of embodiment C9, which comprises shipping thecontainer.

D1. A method for manufacturing 6-hydroxyhexanoic acid, which comprisesculturing an engineered microorganism of any one of embodiments A29,A30, B32 or B33 under culture conditions in which the culturedmicroorganism produces 6-hydroxyhexanoic acid.

D1.1. The method of embodiment D1, wherein the host microorganism fromwhich the engineered microorganism is produced does not produce adetectable amount of 6-hydroxyhexanoic acid.

D2. The method of embodiment D1 or D1.1, wherein the culture conditionscomprise fermentation conditions.

D3. The method of any one of embodiments D1-D2, wherein the cultureconditions comprise introduction of biomass.

D4. The method of D1 or D2, wherein the culture conditions compriseintroduction of glucose.

D5. The method of D1 or D2, wherein the culture conditions compriseintroduction of hexane.

D6. The method of any one of embodiments D1-D5, wherein the6-hydroxyhexanoic acid is produced with a yield of greater than about0.3 grams per gram of glucose added.

D7. The method of any one of embodiments D1-D6, which comprisespurifying the 6-hydroxyhexanoic acid from the cultured microorganisms.

D8. The method of embodiment D7, which comprises modifying the6-hydroxyhexanoic acid, thereby producing modified 6-hydroxyhexanoicacid.

D9. The method of any one of embodiments D1-D8, which comprises placingthe cultured microorganisms, the 6-hydroxyhexanoic acid or the modified6-hydroxyhexanoic acid in a container.

D10. The method of embodiment D9, which comprises shipping thecontainer.

E1. A method for preparing an engineered microorganism that producesadipic acid, which comprises:

-   -   (a) introducing a genetic modification to a host organism that        adds or increases monooxygenase activity, thereby producing        engineered microorganisms having detectable and/or increased        monooxygenase activity; and    -   (b) selecting for engineered microorganisms that produce adipic        acid.

E1.1. The method of embodiment E1, wherein the monooxygenase activity isincorporation of a hydroxyl moiety into a six-carbon molecule.

E1.2. The method of embodiment E1 or E1.1, wherein the six-carbonmolecule is hexanoate.

E2. A method for preparing an engineered microorganism that producesadipic acid, which comprises:

-   -   (a) culturing a host organism with hexane as a nutrient source,        thereby producing engineered microorganisms having detectable        monooxygenase activity; and    -   (b) selecting for engineered microorganisms that produce adipic        acid.

E2.1. The method of embodiment E2, wherein the monooxygenase activity isincorporation of a hydroxyl moiety into a six-carbon molecule.

E2.2. The method of embodiment E2 or E2.1, wherein the six-carbonmolecule is hexanoate.

E3. The method of any one of embodiments E1-E2.2, which comprisesselecting the engineered microorganisms that have a detectable amount ofthe monooxygenase activity.

E4. The method of any one of embodiments E1-E3, which comprisesintroducing a genetic modification that adds or increases a hexanoatesynthase activity, thereby producing engineered microorganisms, andselecting for engineered microorganisms having detectable and/orincreased hexanoate synthase activity.

E5. The method of embodiment E4, wherein the genetic modificationencodes a polypeptide having a hexanoate synthase subunit A activity, ahexanoate synthase subunit B activity, or a hexanoate synthase subunit Aactivity and a hexanoate synthase subunit B activity.

E6. The method of any one of embodiments E1-E5, which comprisesintroducing a genetic modification that adds or increases 6-oxohexanoicacid dehydrogenase activity, thereby producing engineeredmicroorganisms, and selecting for engineered microorganisms havingdetectable and/or increased 6-oxohexanoic acid dehydrogenase activityrelative to the host microorgansim.

E7. The method of any one of embodiments E1-E6, which comprisesintroducing a genetic modification that adds or increases a6-hydroxyhexanoic acid dehydrogenase activity, thereby producingengineered microorganisms, and selecting for engineered microorganismshaving a detectable and/or increased 6-hydroxyhexanoic aciddehydrogenase activity relative to the host microorganism.

E7.1 The method of any one of embodiments E1-E7, which comprisesintroducing a genetic modification that adds or increases a6-hydroxyhexanoic acid dehydrogenase activity, thereby producingengineered microorganisms, and selecting for engineered microorganismshaving a detectable and/or increased omega hydroxyl fatty aciddehydrogenase activity relative to the host microorganism.

E8. The method of any one of embodiments E1-E7.1, which comprisesintroducing a genetic modification that adds or increases a thioesteraseactivity, thereby producing engineered microorganisms, and selecting forengineered microorganisms having a detectable and/or increasedthioesterase activity relative to the host microorganism.

E9. The method of any one of embodiments E1-E8, which comprisesintroducing a genetic modification that reduces 6-hydroxyhexanoic acidconversion, thereby producing engineered microorganisms, and selectingfor engineered microorganisms having reduced 6-hydroxyhexanoic acidconversion relative to the host microorganism.

E10. The method of any one of embodiments E1-E9, which comprisesintroducing a genetic modification that reduces beta-oxidation activity,thereby producing engineered microorganisms, and selecting forengineered microorganisms having reduced beta-oxidation activityrelative to the host microorganism.

E11. The method of any one of embodiments E1-E11, which comprisesintroducing a genetic modification that results in substantial hexanoateusage by the monooxygenase activity, thereby producing engineeredmicroorganisms, and selecting for engineered microorganisms in whichsubstantial hexanoate usage is by the monooxygenase activity relative tothe host microorganism.

F1. A method for preparing a microorganism that produces adipic acid,which comprises: (a) introducing one or more genetic modifications to ahost organism that add or increase one or more activities selected fromthe group consisting of 6-oxohexanoic acid dehydrogenase activity, omegaoxo fatty acid dehydrogenase activity, 6-hydroxyhexanoic aciddehydrogenase activity, omega hydroxyl fatty acid dehydrogenaseactivity, hexanoate synthase activity and monooxygenase activity,thereby producing engineered microorganisms, and (b) selecting forengineered microorganisms that produce adipic acid.

F2. The method of embodiment F1, which comprises selecting forengineered microorganisms having one or more detectable and/or increasedactivities selected from the group consisting of 6-oxohexanoic aciddehydrogenase activity, omega oxo fatty acid dehydrogenase activity,6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity, hexanoate synthase activity and monooxygenaseactivity, relative to the host microorganism.

F3. The method of embodiment F1 or F2, which comprises introducing agenetic modification that reduces 6-hydroxyhexanoic acid conversion,thereby producing engineered microorganisms, and selecting forengineered microorganisms having reduced 6-hydroxyhexanoic acidconversion relative to the host microorganism.

F4. The method of any one of embodiments F1-F3, which comprisesintroducing a genetic modification that reduces beta-oxidation activity,thereby producing engineered microorganisms, and selecting forengineered microorganisms having reduced beta-oxidation activityrelative to the host microorganism.

F5. The method of any one of embodiments F1-F4, which comprisesintroducing a genetic modification that results in substantial hexanoateusage by the monooxygenase activity, thereby producing engineeredmicroorganisms, and selecting for engineered microorganisms in whichsubstantial hexanoate usage is by the monooxygenase activity relative tothe host microorganism.

G1. A method for preparing a microorganism that produces6-hydroxyhexanoic acid, which comprises: (a) introducing one or moregenetic modifications to a host organism that add or increase one ormore activities selected from the group consisting of 6-oxohexanoic aciddehydrogenase activity, hexanoate synthase activity and monooxygenaseactivity, thereby producing engineered microorganisms, (b) introducing agenetic modification to the host organism that reduces 6-hydroxyhexanoicacid conversion, and (c) selecting for engineered microorganisms thatproduce 6-hydroxyhexanoic acid.

G2. The method of embodiment G1, which comprises selecting forengineered microorganisms having reduced 6-hydroxyhexanoic acidconversion relative to the host microorganism.

G3. The method of embodiment G1 or G2, which comprises selecting forengineered microorganisms having one or more detectable and/or increasedactivities selected from the group consisting of 6-oxohexanoic aciddehydrogenase activity, omega oxo fatty acid dehydrogenase activity,6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity, hexanoate synthase activity and monooxygenaseactivity, relative to the host microorganism.

G4. The method of any one of embodiments G1-G3, which comprisesintroducing a genetic modification that reduces beta-oxidation activity,thereby producing engineered microorganisms, and selecting forengineered microorganisms having reduced beta-oxidation activityrelative to the host microorganism.

G5. The method of any one of embodiments G1-G4, which comprisesintroducing a genetic modification that results in substantial hexanoateusage by the monooxygenase activity, thereby producing engineeredmicroorganisms, and selecting for engineered microorganisms in whichsubstantial hexanoate usage is by the monooxygenase activity relative tothe host microorganism.

H1. A method, comprising:

-   -   contacting an engineered microorganism with an feedstock        comprising one or more polysaccharides, wherein the engineered        microorganism comprises:    -   a. a genetic alteration that blocks beta oxidation activity, and    -   b. a genetic alteration that adds or increases a monooxygenase        activity or a genetic alteration that adds or increases        hexanoate synthetase activity; and    -   culturing the engineered microorganism under conditions in which        adipic acid is produced.

H1.1. The method of embodiment H1, wherein the engineered microorganismcomprises a genetic alteration that adds or increases hexanoatesynthetase activity.

H1.2. The method of embodiment H1.1, wherein the engineeredmicroorganism comprises a heterologous polynucleotide encoding apolypeptide having hexanoate synthase subunit A activity.

H1.3. The method of embodiment H1.1, wherein the engineeredmicroorganism comprises a heterologous polynucleotide encoding apolypeptide having hexanoate synthase subunit B activity.

H1.4. The method of embodiment H1.2 or H1.3, wherein the heterologouspolynucleotide independently is selected from a bacterium.

H1.5. The method of embodiment H1.4, wherein the bacterium is a Bacillusbacterium.

H1.6. The method of embodiment H1.5, wherein the Bacillus bacterium isB. megaterium.

H2. The method of any one of embodiment H1 or H1.6, wherein themicroorganism is a Candida yeast.

H3. The method of embodiment H2, wherein the microorganism is a C.tropicalis strain.

H4. The method of any one of embodiments H1 to H3, wherein the geneticalteration that increases monooxygenase activity comprises a geneticalteration that increases cytochrome P450 reductase activity.

H5. The method of embodiment H4, wherein the genetic alterationincreases the number of copies of a polynucleotide that encodes apolypeptide having the cytochrome P450 reductase activity.

H6. The method of embodiment H4, wherein the genetic alteration places apromoter in functional connection with a polynucleotide that encodes apolypeptide having the cytochrome P450 reductase activity.

H7. The method of any one of embodiments H1 to H6, wherein themonooxygenase activity is a CYP52A15 activity, CYP52A16 activity, or aCYP52A15 activity and CYP52A16 activity.

H8. The method of any one of embodiments H1 to H7, wherein the geneticalteration increases the number of copies of a polynucleotide thatencodes a polypeptide having the monooxygenase activity.

H9. The method of any one of embodiments H1 to H7, wherein the geneticalteration places a promoter in functional connection with apolynucleotide that encodes a polypeptide having the monooxygenaseactivity.

H10. The method of any one of embodiments H1 to H7, wherein the geneticalteration that blocks beta oxidation activity disrupts acyl-CoA oxidaseactivity.

H11. The method of embodiment H10, wherein the genetic alterationdisrupts POX4 and POX5 activity.

H12. The method of embodiment H10 or H11, wherein the genetic alterationdisrupts a polynucleotide that encodes a polypeptide having the acyl-CoAoxidase activity.

H13. The method of embodiment H10 or H11, wherein the genetic alterationdisrupts a promoter in functional connection with a polynucleotide thatencodes a polypeptide having the acyl-CoA oxidase activity.

H14. The method of any one of embodiments H1 to H13, wherein thefeedstock comprises a 6-carbon sugar.

H15. The method of any one of embodiments H1 to H13, wherein thefeedstock comprises a 5-carbon sugar.

H16. The method of any one of embodiments H1 to H15, wherein the adipicacid is produced at a level of about 80% or more of theoretical yield.

H17. The method of any one of embodiments H1 to H16, comprisingdetecting the amount of adipic acid produced.

H18. The method of any one of embodiments H1 to H17, comprisingisolating the adipic acid produced.

H19. The method of any one of embodiments H1 to H18, wherein the cultureconditions comprise fermenting the engineered microorganism.

I1. A method, comprising:

-   -   contacting an engineered microorganism with a feedstock        comprising one or more paraffins, wherein the engineered        microorganism comprises a genetic alteration that partially        blocks beta oxidation activity; and    -   culturing the engineered microorganism under conditions in which        adipic acid is produced.

I1.1. The method of embodiment I1, wherein the microorganism comprises agenetic alteration that increases a monooxygenase activity.

I2. The method of embodiment I1 or I1.1, wherein the microorganism is aCandida yeast.

I3. The method of embodiment I2, wherein the microorganism is a C.tropicalis strain.

I4. The method of any one of embodiments I1 to I3, wherein the geneticalteration that increases monooxygenase activity comprises a geneticalteration that increases cytochrome P450 reductase activity.

I5. The method of embodiment I4, wherein the genetic alterationincreases the number of copies of a polynucleotide that encodes apolypeptide having the cytochrome P450 reductase activity.

I6. The method of embodiment I4, wherein the genetic alteration places apromoter in functional connection with a polynucleotide that encodes apolypeptide having the cytochrome P450 reductase activity.

I7. The method of any one of embodiments I1 to I6, wherein themonooxygenase activity is a CYP52A15 activity, CYP52A16 activity, or aCYP52A15 activity and CYP52A16 activity.

I8. The method of any one of embodiments I1 to I7, wherein the geneticalteration increases the number of copies of a polynucleotide thatencodes a polypeptide having the monooxygenase activity.

I9. The method of any one of embodiments I1 to I7, wherein the geneticalteration places a promoter in functional connection with apolynucleotide that encodes a polypeptide having the monooxygenaseactivity.

I10. The method of any one of embodiments I1 to I7, wherein the geneticalteration that blocks beta oxidation activity disrupts acyl-CoA oxidaseactivity.

I11. The method of embodiment I10, wherein the genetic alterationdisrupts POX4 or POX5 activity.

I12. The method of embodiment I10 or I11, wherein the genetic alterationdisrupts a polynucleotide that encodes a polypeptide having the acyl-CoAoxidase activity.

I13. The method of embodiment I10 or I11, wherein the genetic alterationdisrupts a promoter in functional connection with a polynucleotide thatencodes a polypeptide having the acyl-CoA oxidase activity.

I14. The method of any one of embodiments I1 to I13, wherein the adipicacid is produced at a level of about 80% or more of theoretical yield.

I15. The method of any one of embodiments I1 to I14, comprisingdetecting the amount of adipic acid produced.

I16. The method of any one of embodiments I1 to I15, comprisingisolating the adipic acid produced.

I17. The method of any one of embodiments I1 to I16, wherein the cultureconditions comprise fermenting the engineered microorganism.

I18. The method of any one of embodiments I1 to I17, wherein theparaffin is a saturated paraffin, unsaturated paraffin, substitutedparaffin, branched paraffin, linear paraffin or combination thereof.

I19. The method of any one of embodiments I1 to I18, wherein theparaffin includes about 1 to about 60 carbon atoms.

I20. The method of any one of embodiments I1 to I19, wherein theparaffin is in a mixture of paraffins.

I21. The method of embodiment I20, wherein the paraffins in the mixtureof paraffins have a mean number of carbon atoms of about 8 to about 18carbon atoms.

I22. The method of embodiment I21, wherein the mean number of carbonatoms is about 10 to about 16 carbon atoms.

I23. The method of embodiment I22, wherein the mean number of carbonatoms is about 12 atoms.

I24. The method of any one of embodiments I1 to I23, wherein theparaffin is in a wax.

I25. The method of any one of embodiments I1 to I23, wherein theparaffin is in an oil.

I26. The method of any one of embodiments I1 to I25, wherein theparaffin is from a petroleum product.

I27. The method of embodiment I26, wherein the petroleum product is apetroleum distillate.

I28. The method of any one of embodiments I1 to I27, wherein theparaffin is from a plant or plant product.

J1. An isolated polynucleotide selected from the group consisting of:

-   -   a polynucleotide having a nucleotide sequence 96% or more        identical to the nucleotide sequence of SEQ ID NO: 1;    -   a polynucleotide having a nucleotide sequence that encodes a        polypeptide of SEQ ID NO: 8; and    -   a polynucleotide having a portion of a nucleotide sequence 96%        or more identical to the nucleotide sequence of SEQ ID NO: 1 and        encodes a polypeptide having fatty alcohol oxidase activity.

J2. An isolated polynucleotide selected from the group consisting of:

-   -   a polynucleotide having a nucleotide sequence 98% or more        identical to the nucleotide sequence of SEQ ID NO: 2;        -   a polynucleotide having a nucleotide sequence that encodes a            polypeptide of SEQ ID NO: 10; and    -   a polynucleotide having a portion of a nucleotide sequence 98%        or more identical to the nucleotide sequence of SEQ ID NO: 2 and        encodes a polypeptide having fatty alcohol oxidase activity.

J3. An isolated polynucleotide selected from the group consisting of:

-   -   a polynucleotide having a nucleotide sequence 95% or more        identical to the nucleotide sequence of SEQ ID NO: 3;    -   a polynucleotide having a nucleotide sequence that encodes a        polypeptide of SEQ ID NO: 9; and    -   a polynucleotide having a portion of a nucleotide sequence 95%        or more identical to the nucleotide sequence of SEQ ID NO: 3 and        encodes a polypeptide having fatty alcohol oxidase activity.

J3.1. An isolated polynucleotide selected from the group consisting of:

-   -   a polynucleotide having a nucleotide sequence 83% or more        identical to the nucleotide sequence of SEQ ID NO: 4;    -   a polynucleotide having a nucleotide sequence that encodes a        polypeptide of SEQ ID NO: 11; and    -   a polynucleotide having a portion of a nucleotide sequence 83%        or more identical to the nucleotide sequence of SEQ ID NO: 4 and        encodes a polypeptide having fatty alcohol oxidase activity.

J3.2. An isolated polynucleotide selected from the group consisting of:

-   -   a polynucleotide having a nucleotide sequence 82% or more        identical to the nucleotide sequence of SEQ ID NO: 5;    -   a polynucleotide having a nucleotide sequence that encodes a        polypeptide of SEQ ID NO: 12; and    -   a polynucleotide having a portion of a nucleotide sequence 82%        or more identical to the nucleotide sequence of SEQ ID NO: 5 and        encodes a polypeptide having fatty alcohol oxidase activity.

J4. An expression vector comprising a polynucleotide of any one ofembodiments J1 to J3.2.

J5. An integration vector comprising a polynucleotide of any one ofembodiments J1 to J3.2.

J6. A microorganism comprising an expression vector of embodiment J4 oran integration vector of embodiment J5.

J7. A culture comprising a microorganism of embodiment J6.

J8. A fermentation device comprising a microorganism of embodiment J7.

J10. A polypeptide encoded by a polynucleotide of any one of embodimentsJ1 to J3 or produced by an expression vector of embodiment J4 ormicroorganism of embodiment J6.

J11. An antibody that specifically binds to a polypeptide of embodimentJ10.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the claimedtechnology. The term “a” or “an” can refer to one of or a plurality ofthe elements it modifies (e.g., “a reagent” can mean one or morereagents) unless it is contextually clear either one of the elements ormore than one of the elements is described. The term “about” as usedherein refers to a value within 10% of the underlying parameter (i.e.,plus or minus 10%), and use of the term “about” at the beginning of astring of values modifies each of the values (i.e., “about 1, 2 and 3”refers to about 1, about 2 and about 3). For example, a weight of “about100 grams” can include weights between 90 grams and 110 grams. Further,when a listing of values is described herein (e.g., about 50%, 60%, 70%,80%, 85% or 86%) the listing includes all intermediate and fractionalvalues thereof (e.g., 54%, 85.4%). Thus, it should be understood thatalthough the present technology has been specifically disclosed byrepresentative embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and such modifications and variations are consideredwithin the scope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

1. A method for producing adipic acid, comprising: (a) contacting aCandida yeast with a feedstock comprising a vegetable oil, wherein theCandida yeast comprises genetic alterations that: (i) partially blockbeta oxidation activity, (ii) add or increase a CYP52A15, CYP52A16, orCYP52A19 monooxygenase activity, and (iii) add or increase amonooxygenase reductase activity; and (b) culturing the Candida yeastunder conditions in which adipic acid is produced at a level of at least0.4 grams per liter of culture medium.
 2. The method of claim 1, whereinthe vegetable oil is a palm oil.
 3. The method of claim 1, wherein thevegetable oil is a soybean oil.
 4. The method of claim 1, wherein thevegetable oil is a fatty acid soap stock.
 5. The method of claim 1,wherein the Candida yeast comprises a genetic alteration that reduces oreliminates the amount of a polypeptide having an acyl-CoA oxidaseactivity.
 6. The method of claim 5, wherein the polypeptide is a POX4polypeptide or a POX5 polypeptide.
 7. The method of claim 6, wherein thepolypeptide is a POX4 polypeptide.
 8. The method of claim 1, wherein theCandida yeast is a genetically modified ATCC20336 yeast or a geneticallymodified ATCC20962 yeast.
 9. The method of claim 1, wherein the geneticalteration that adds or increases the monooxygenase reductase activitycomprises (i) increasing the copy number of a CPRA coding sequence, or(ii) modifying or adding a promoter in operable connection with a CPRAcoding sequence.
 10. The method of claim 1, wherein the geneticalteration that adds or increases the monooxygenase reductase activitycomprises (i) increasing the copy number of a CPRB coding sequence, or(ii) modifying or adding a promoter in operable connection with a CPRBcoding sequence.
 11. The method of claim 1, wherein the geneticalteration that adds or increases the monooxygenase reductase activitycomprises (i) increasing the copy number of a CPRA coding sequence, or(ii) modifying or adding a promoter in operable connection with a CPRAcoding sequence; and (iii) increasing the copy number of a CPRB codingsequence, or (iv) modifying or adding a promoter in operable connectionwith a CPRB coding sequence.
 12. The method of claim 1, wherein theCandida yeast comprises a genetic alteration that adds or increases aCYP52A15 monooxygenase activity.
 13. The method of claim 12, wherein thegenetic alteration increases the copy number of a polynucleotide thatencodes a polypeptide having the CYP52A15 monooxygenase activity. 14.The method of claim 12, wherein the genetic alteration comprisesmodifying or adding a promoter in operable connection with apolynucleotide that encodes a polypeptide having the CYP52A15monooxygenase activity.
 15. The method of claim 1, wherein the Candidayeast comprises a genetic alteration that adds or increases a CYP52A16monooxygenase activity.
 16. The method of claim 15, wherein the geneticalteration increases the copy number of a polynucleotide that encodes apolypeptide having the CYP52A16 monooxygenase activity.
 17. The methodof claim 15, wherein the genetic alteration comprises modifying oradding a promoter in operable connection with a polynucleotide thatencodes a polypeptide having the CYP52A16 monooxygenase activity. 18.The method of claim 1, wherein the Candida yeast comprises a geneticalteration that adds or increases a CYP52A19 monooxygenase activity. 19.The method of claim 18, wherein the genetic alteration increases thecopy number of a polynucleotide that encodes a polypeptide having theCYP52A19 monooxygenase activity.
 20. The method of claim 18, wherein thegenetic alteration comprises modifying or adding a promoter in operableconnection with a polynucleotide that encodes a polypeptide having theCYP52A19 monooxygenase activity.